Methods and compositions for prolonging elimination half-times of bioactive compounds

ABSTRACT

Peptide ligands having affinity for IgG or for serum albumin are disclosed. Also disclosed are hybrid molecules comprising a peptide ligand domain and an active domain. The active domain may comprise any molecule having utility as a therapeutic or diagnostic agent. The hybrid molecules of the invention may be prepared using any of a number techniques including production in and purification from recombinant organisms transformed or transfected with an isolated nucleic acid encoding the hybrid molecule, or by chemical synthesis of the hybrid. The hybrid molecules have utility as agents to alter the elimination half-times of active domain molecules. Elimination half-time is altered by generating a hybrid molecule of the present invention wherein the peptide ligand has binding affinity for a plasma protein. In a preferred embodiment, a bioactive molecule having a short elimination half-time is incorporated as or into an active domain of the hybrid molecules of the invention, and the binding affinity of the peptide ligand domain prolongs the elimination half-time of the hybrid as compared to that of the bioactive molecule.

FIELD OF THE INVENTION

[0001] This invention relates to novel compounds termed peptide ligands which bind a predetermined molecule such as a plasma protein. In particular aspects, the invention relates to compositions comprising a hybrid molecule comprising a peptide ligand domain and an active domain such as a biologically active molecule. The active domain may comprise a molecule useful for diagnostic or therapeutic purposes. In preferred embodiments, the hybrid compositions comprising the peptide ligand domain and active domain have improved pharmacokinetic or pharmacological properties. The invention further provides for the research, diagnostic and therapeutic use of the peptide ligand and includes compositions such as pharmaceutical compositions comprising the peptide ligand molecules.

DESCRIPTION OF RELATED DISCLOSURES

[0002] Phage-display provides a means for generating constrained and unconstrained peptide libraries (Devlin et al., (1990) Science 249:404-406; Cwirla et al., (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382; Lowman (1997) Ann. Rev. Biophys. Biomol. Struct. 26:401-424). These libraries can be used to identify and select peptide ligands that can bind a predetermined target molecule (Lowman (1997), supra); Clackson and Wells (1994) Trends Biotechnol. 12:173-184; Devlin et al., (1990) supra). The technique has been used to identify peptide motifs that home to a cellular target (Arap et al., (1998) Science 279:377-380); bind the human type I interleukin 1 (IL-1) receptor blocking the binding of IL-α (Yanofsky et al., (1996) Proc. Natl. Acad. Sci. USA 93:7381-7386); bind to and activate the receptor for the cytokine erythropoietin (EPO) (Wrighton et al., (1996) Science 273:458-463); bind the human thrombopoietin receptor and compete with the binding of the natural ligand thrombopoietin (TPO) (Cwirla et al., (1996) Science 276:1696-1699), or to generate affinity improved or matured peptide ligands from native protein binding ligands (Lowman et al., (1991) Biochemistry 30:10832-10838).

[0003] Using structurally constrained peptide libraries generated by monovalent phage display, 14 amino acid peptides that specifically bind to insulin-like growth factor 1 binding proteins (IGFBPs) have been isolated (Lowman et al. (1998), Biochemistry 37:8870-8878). The peptides contain a helix structure and bind IGFBPs in vitro liberating insulin like growth factor-a (IGF-1) activity (Lowman et al., (1998) supra). Utilizing in vivo phage selection peptides capable of mediating selective localization to various organs such as brain and kidney (Pasqualini and Ruoslohti (1996) Nature 380:364-366) as well as peptides that home to particular tumor types bearing α_(V)β₃ or αVβ₅ integrins have been identified (Arap et al. (1998), Science 279:377-380). U.S. Pat. No. 5,627,263 describes peptides that are recognized by and selectively bind the α₅β₁ integrin. Examples of affinity or specificity improved proteins include human growth hormone, zinc fingers, protease inhibitors, atrial natriuretic factor, and antibodies (Wells, J. and Lowman H. (1992), Curr. Opin. Struct. Biol. 2:597-604; Clackson, T. and Wells, J. (1994), Trends Biotechnol. 12:173-184; Lowman et al., (1991) Biochemistry 30(10):832-838; Lowman and Wells J. (1993), J. Mol. Biol. 234:564-578; Dennis M. and Lazarus R. (1994), J. Biol. Chem. 269(22):137-144).

[0004] It has been suggested that the pharmakodynamics of insulin are altered if bound to serum albumin. Acylation of insulin with saturated fatty acids containing 10-16 carbon atoms produces insulin with affinity for albumin (Kurtzhals, P. et al. (1995) Biochem. J. 312:725-731). Differences in albumin binding affinity among acylated insulins were correlated with the timing of the blood-glucose lowering effects of the various molecules after subcutaneous injection into rabbits. Tighter binding to albumin was correlated with a delay in blood glucose lowering, possibly due to acylated insulin binding albumin in the subcutaneous tissue, resulting in a lower absorption rate of the acylated insulins when compared with non-acylated insulin.

[0005] A serum albumin-CD4 conjugate in which the V1 and V2 domains of CD4 were fused with human serum albumin (HSA) has been described (Yeh, P. et al. (1992), Proc. Natl. Acad. Sci. USA 89:1904-1908). The conjugate's elimination half-time was 140-fold that of a soluble CD4 (sCD4) in a rabbit experimental model.

[0006] Extended in vivo half-times of human soluble complement receptor type 1 (sCR1) fused to the albumin binding domains from Streptococcal protein G have been reported (Makrides, S. et al. (1996) J. Pharmacol. Exptl. Ther. 277:532-541). The constructs contained albumin binding domains of protein G having approximately 80 amino acids (fragment BA), and approximately 155 amino acids (fragment BABA).

[0007] The pharmacokinetics of a labeled IgG binding domain derived from the Z domain of protein A having approximately 60 amino acids and of a serum albumin binding domain derived from Streptococcal protein G (B-domain) having approximately 200 amino acids have been described (EP 0 486,525).

SUMMARY OF THE INVENTION

[0008] The present invention provides novel compounds that bind to plasma proteins. The compounds of the present invention (referred to as peptide ligands) are, for example, peptides or peptide derivatives such as peptide mimetics and peptide analogs. According to preferred aspects of the invention, the compounds are non-naturally occurring amino acid sequences that bind plasma proteins such as serum albumin or a portion of an immunoglobulin, as for example, IgG-Fc. Preferably the peptide ligand is a non-naturally occurring amino acid sequence of between about 10 and 20 amino acid residues.

[0009] Such compounds preferably bind a desired plasma protein with an affinity characterized by a dissociation constant, K_(d), that is less than about 100 μM, preferably less than about 100 nM, and preferably do not substantially bind other plasma proteins. Specific examples of such compounds include linear or cyclic, especially cyclic peptides, preferably between about 10 and 20 amino acid residues in length, and combinations thereof, optionally modified at the N-terminus or C-terminus or both, as well as their salts and derivatives, functional analogues thereof and extended peptide chains carrying amino acids or polypeptides at the termini of the sequences.

[0010] Preferred peptide ligands bind IgG-Fc and include linear and cyclic peptides, preferably cyclic peptide compounds comprising the following core formula:

[0011] Xaa_(i)-Cys-Xaa_(j)-Cys-Xaa_(k), wherein Xaa_(i) is absent or is a peptide of between 1 and 4 amino acids, preferably 4 amino acids; X_(j) is preferably 9 amino acids having a preferred sequence Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Leu-Val-Trp (SEQ ID NO: 9); or Xaa₁-Xaa₂-Xaa₃-Xaa₄-Gly-Glu-Leu-Val-Trp (SEQ ID NO: 10); or Xaa₁-Xaa₂-Xaa₃-Xaa₄-Gly-Glu-Leu-Val-Trp (SEQ ID NO: 10), wherein Xaa₁ preferably is Ala, Ser, or Thr; Xaa₂ preferably is Trp or Tyr, Xaa₃ preferably is His, or Trp; Xaa₄ preferably is Leu or Met, and Xaa_(k) is absent or between 1 and 5 amino acids, preferably 5 amino acids, so long as the cyclic peptide or analog thereof retains the qualitative biological activity of IgG-Fc binding.

[0012] Preferred among this group of compounds are compounds that bind IgG-Fc comprising the sequence: Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Leu-Val-Trp-Cys-Xaa-Xaa-Xaa- (SEQ ID NO: 11) Xaa-Xaa; Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Gly-Glu-Leu-Val-Trp-Cys-Xaa-Xaa-Xaa- (SEQ ID NO: 12) Xaa-Xaa; Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Gly-Glu-Leu-Val-Trp-Cys-Xaa₉- (SEQ ID NO: 13) Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃, wherein Xaa₅ is Ala, Ser, or Thr; Xaa₆ is Trp or Tyr; Xaa₇ is His or Trp; and Xaa₈ is Leu or Met; and Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Gly-Glu-Leu-Val-Trp-Cys-Xaa₉- (SEQ ID NO: 14) Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃ wherein Xaa₄ is Ser, Arg, or Asp; Xaa₅ is Ala, Ser, or Thr; Xaa₆ is Trp or Tyr; Xaa₇ is His or Trp; Xaa₈ is Leu or Met; and Xaa₉ is Glu, Ser, Thr or Val.

[0013] Preferred peptide ligands that bind serum albumin include linear and cyclic peptides, preferably cyclic peptide compounds comprising the following formulae: (Xaa)_(x)-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-(Xaa)_(z) (Xaa)_(x)-Phe-Cys-Xaa-Asp-Trp-Pro-Xaa-Xaa-Xaa-Ser-Cys-(Xaa)_(z) (SEQ ID NO: 113) (Xaa)_(x)-Val-Cys-Tyr-Xaa-Xaa-Xaa-Ile-Cys-Phe-(Xaa)_(z) (SEQ ID NO: 114) (Xaa)_(x)-Cys-Tyr-Xaa₁-Pro-Gly-Xaa-Cys-(Xaa)_(z) (SEQ ID NO: 115) and (Xaa)_(x)-Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp-(Xaa)_(z) (SEQ ID NO: 116)

[0014] Preferred are peptide compounds of the general formulae wherein Xaa is an amino acid and x and z are a whole number greater or equal to 0 (zero), generally less than 100, preferably less than 10 and more preferably 0, 1, 2, 3, 4 or 5 and more preferably 4 or 5 and Xaa₁ is selected from the group consisting of Ile, Phe, Tyr and Val.

[0015] In particular aspects the invention is directed to combinations of a peptide ligand with a bioactive compound to form a hybrid molecule that comprises a peptide ligand domain and an active domain. The bioactive compounds of the invention include any compound useful as a therapeutic or diagnostic agent. Non-limiting examples of bioactive compounds include polypeptides such as enzymes, hormones, cytokines, antibodies or antibody fragments, as well as organic compounds such as analgesics, antipyretics, antiinflammatory agents, antibiotics, antiviral agents, anti-fungal drugs, cardiovascular drugs, drugs that affect renal function and electrolyte metabolism, drugs that act on the central nervous system and chemotherapeutic drugs, to name but a few.

[0016] In preferred embodiments, the hybrid molecules comprising a peptide ligand domain and an active domain have improved pharmacokinetic or pharmacodynamic properties as compared to the same bioactive molecule comprising the active domain but lacking the peptide ligand domain. The improved pharmacokinetic or pharmacodynamic properties of the hybrids thereby provide for low-dose pharmaceutical formulations and novel pharmaceutical compositions. In certain aspects, the invention provides for methods of using the novel compositions including the therapeutic or diagnostic use of the hybrid molecules.

[0017] In particular aspects, the invention is directed to combinations of peptide ligands with bioactive compounds that have relatively short elimination half-times. The combinations are prepared with various objectives in mind, including improving the therapeutic or diagnostic efficacy of the bioactive compound in aspects of the invention involving in vivo use of the bioactive compound, by for example, increasing the elimination half-time of the bioactive compound. Fusing or linking (i.e., “conjugating”) the peptide ligand directed against a plasma protein such as serum albumin, an immunoglobulin, an apolipoprotein or transferrin to a bioactive compound provides compositions with increased elimination half-times. Such combinations or fusions are conveniently made in recombinant host cells, or by the use of bifunctional crosslinking agents.

[0018] Other aspects of the invention include methods and compositions to purify antibodies using peptide ligands having binding affinity for immunoglobulins, such as, for example, the IgG-Fc peptide ligands disclosed herein.

[0019] The present invention further extends to therapeutic and diagnostic applications for the compositions described herein. Therefore, the invention includes pharmaceutical compositions comprising a pharmaceutically acceptable excipient and the hybrid molecules of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1. Phage competitive ELISA assay showing IgG binding of peptide-ligand tagged anti-VEGF Fab-phagemid particles. Four different constructs are shown: pY0192-569 (large filled circles), pY0192-570 (large open circles), PY0317-569 (small filled circles), and pY0317-570 (“x”'s).

[0021]FIG. 2. BIAcore™ analysis of IgG binding to peptide-ligand tagged anti-VEGF Fab Y0317-570 (tagged; top panel) Y0317 Fab (control; bottom panel). A cartoon illustration at top shows a model for the binding events observed in the tagged Fab experiment.

[0022]FIG. 3. Group average serum concentration vs. time data (±SD) are presented in the figure for Fab-Y0317-570 and Fab-Y0317.

[0023]FIG. 4. The peptide sequences displayed by phage clones selected for binding to rabbit, human or rat albumin are shown in FIG. 4. Also indicated is the ability of individual phage clones to bind the 3 species of immobilized albumin.

[0024]FIGS. 5A and 5B. Sequences identified following soft randomization are shown in FIG. 5 along with their species specificity as determined by phage ELISA.

[0025]FIG. 6. Clones originating from the RB soft randomization library were found by ELISA to bind each of these species of albumin and were specific for albumin based upon their lack of binding to ovalbumin and casein.

[0026]FIG. 7. Clones that bind to multiple species of albumin (multi-species binders) are listed in FIG. 7.

[0027]FIGS. 8A, 8B and 8C. Sequences from libraries selected against rat, rabbit and human albumin are shown in FIGS. 8A, 8B, and 8C respectively.

[0028]FIG. 9. Peptides corresponding to identified phage sequences were synthesized and their affinity for rat, rabbit or mouse albumin measured using the SA08b binding assay.

[0029]FIG. 10A peptides corresponding to the SA06 identified phage sequence was synthesized and its affinity for rat, rabbit or mouse albumin measured using the SA08b binding assay.

[0030]FIG. 11. The SA06 sequence was added to the carboxy terminus of either the light chain (D3H44-L) or heavy chain (D3H44-Ls) of the Fab. In addition, identical constructs were made with the intra-chain disulfide replaced by alanines (D3H44-Ls and D3H44-Hs, respectively) as depicted in FIG. 11.

[0031]FIG. 12. Purified D3H44 fusions retained their ability to bind TF as measured using a FX activation assay.

[0032]FIG. 13. Purified D3H44 fusions retained their ability to bind TF as measured using a prothrombin time assay that measures prolongation of tissue factor dependent clotting.

[0033]FIG. 14 Unlike D3H44 lacking the albumin binding sequence (WT), both D3H44-L and D3H44-Ls are able to bind to albumin as measured in the SA08b binding assay.

[0034]FIG. 15 Both D3H44 albumin-binding fusions are capable of binding TF and albumin simultaneously as judged by a biotin-TF binding assay.

[0035]FIG. 16 Fusion of the albumin binding peptide to D3H44 results in a protein having improved pharmacokinetic parameters.

[0036]FIG. 17. Fusion of the albumin binding peptide to D3H44 results in a protein having improved pharmacokinetic parameters.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] I. Definitions

[0038] The term “peptide ligand” within the context of the present invention is meant to refer to non-naturally occurring amino acid sequences that function to bind a particular target molecule. Peptide ligands within the context of the present invention are generally constrained (that is, having some element of structure as, for example, the presence of amino acids which initiate a β turn or β pleated sheet, or for example, cyclized by the presence of disulfide-bonded Cys residues) or unconstrained (linear) amino acid sequences of less than about 50 amino acid residues, and preferably less than about 40 amino acids residues. Of the peptide ligands less than about 40 amino acid residues, preferred are the peptide ligands of between about 10 and about 30 amino acid residues and especially the peptide ligands of about 20 amino acid residues. However, upon reading the instant disclosure, the skilled artisan will recognize that it is not the length of a particular peptide ligand but its ability to bind a particular target molecule that distinguishes the peptide ligand of the present invention. Therefore peptide ligands of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25 amino acid residues, for example, are equally likely to be peptide ligands within the context of the present invention.

[0039] A peptide ligand of the present invention will bind a target molecule with sufficient affinity and specificity if the peptide ligand “homes” to, “binds” or “targets” a target molecule such as a specific cell type bearing the target molecule in vitro and preferably in vivo (see, for example, the use of the term “homes to,” “homing,” and “targets” in Pasqualini and Ruoslahti (1996) Nature, 380:364-366 and Arap et al., (1998) Science, 279:377-380). In general, the peptide ligand will bind a target molecule with an affinity characterized by a dissociation constant, K_(d), of less than about 1 μM, preferably less than about 100 nM and more preferably less than about 10 nM. However, peptide ligands having an affinity for a target molecule of less than about 1 nM and preferably between about 1 pM and 1 nM are equally likely to be peptide ligands within the context of the present invention. In general a peptide ligand that binds a particular target molecule as described above can be isolated and identified by any of a number of art-standard techniques as described herein.

[0040] Peptides ligands are amino acid sequences as described above which may contain naturally as well as non-naturally occurring amino acid residues. Therefore, so-called “peptide mimetics” and “peptide analogs” which may include non-amino acid chemical structures that mimic the structure of a particular amino acid or peptide may be peptide ligands within the context of the invention. Such mimetics or analogs are characterized generally as exhibiting similar physical characteristics such as size, charge or hydrophobicity present in the appropriate spacial orientation as found in their peptide counterparts. A specific example of a peptide mimetic compound is a compound in which the amide bond between one or more of the amino acids is replaced by, for example, a carbon-carbon bond or other bond as is well known in the art (see, for example Sawyer, in Peptide Based Drug Design pp. 378-422 (ACS, Washington D.C. 1995)).

[0041] Therefore, the term “amino acid” within the scope of the present invention is used in its broadest sense and is meant to include naturally occurring L α-amino acids or residues. The commonly used one and three letter abbreviations for naturally occurring amino acids are used herein (Lehninger, A. L., Biochemistry, 2d ed., pp. 71-92, (1975), Worth Publishers, New York). The correspondence between the standard single letter codes and the standard three letter codes is well known to the skilled artisan, and is reproduced here: A=Ala; C=Cys; D=Asp; E=Glu; F Phe; G=Gly; H His; I=Ile; K=Lys; L=Leu; M=Met; N=Asn; P=Pro; Q=Gln; R=Arg; S=Ser; T=Thr, V=Val; W=Trp; Y=Tyr. The term includes D-amino acids as well as chemically modified amino acids such as amino acid analogs, naturally occurring amino acids that are not usually incorporated into proteins such as norleucine, and chemically synthesized compounds having properties known in the art to be characteristic of an amino acid. For example, analogs or mimetics of phenylalanine or proline, which allow the same conformational restriction of the peptide compounds as natural Phe or Pro are included within the definition of amino acid. Such analogs and mimetics are referred to herein as “functional equivalents” of an amino acid. Other examples of amino acids are listed by Roberts and Vellaccio The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, eds., Vol. 5 p. 341, Academic Press, Inc., N.Y. 1983, which is incorporated herein by reference.

[0042] Peptide ligands synthesized by, for example, standard solid phase synthesis techniques, are not limited to amino acids encoded by genes. Commonly encountered amino acids which are not encoded by the genetic code, include, for example, those described in International Publication No. WO 90/01940 such as, for example, 2-amino adipic acid (Aad) for Glu and Asp; 2-aminopimelic acid (Apm) for Glu and Asp; 2-aminobutyric (Abu) acid for Met, Leu, and other aliphatic amino acids; 2-aminoheptanoic acid (Ahe) for Met, Leu and other aliphatic amino acids; 2-aminoisobutyric acid (Aib) for Gly; cyclohexylalanine (Cha) for Val, and Leu and Ile; homoarginine (Har) for Arg and Lys; 2,3-diaminopropionic acid (Dpr) for Lys, Arg and His; N-ethylglycine (EtGly) for Gly, Pro, and Ala; N-ethylglycine (EtGly) for Gly, Pro, and Ala; N-ethylasparigine (tAsn) for Asn, and Gln; Hydroxyllysine (Hyl) for Lys; allohydroxyllysine (AHyl) for Lys; 3-(and 4)-hydoxyproline (3Hyp, 4Hyp) for Pro, Ser, and Thr; allo-isoleucine (AIle) for Ile, Leu, and Val; p-amidinophenylalanine for Ala; N-methylglycine (MeGly, sarcosine) for Gly, Pro, and Ala; N-methylisoleucine (MeIle) for Ile; Norvaline (Nva) for Met and other aliphatic amino acids; Norleucine (Nle) for Met and other aliphatic amino acids; Ornithine (Orn) for Lys, Arg and His; Citrulline (Cit) and methionine sulfoxide (MSO) for Thr, Asn and Gln; N-methylphenylalanine (MePhe), trimethylphenylalanine, halo (F, Cl, Br, and I) phenylalanine, trifluorylphenylalanine, for Phe.

[0043] Peptide ligands within the context of the present invention may be “engineered”, i.e., they are non-native or non-naturally occurring peptide ligands. By “non-native” or “non-naturally occurring” is meant that the amino acid sequence of the particular peptide ligand is not found in nature. That is to say, amino acid sequences of non-native or non-naturally occurring peptide ligands do not correspond to an amino acid sequence of a naturally occurring protein or polypeptide. Peptide ligands of this variety may be produced or selected using a variety of techniques well known to the skilled artisan. For example, constrained or unconstrained peptide libraries may be randomly generated and displayed on phage utilizing art standard techniques, for example, Lowman et al., (1998) Biochemistry 37:8870-8878.

[0044] Peptide ligands, when used within the context of the present invention, may be “conjugated” to a therapeutic or diagnostic substance. The term “conjugated” is used in its broadest sense to encompass all methods of attachment or joining that are known in the art. For example, in a typical embodiment, the therapeutic or diagnostic substance is a protein (referred to herein as a “protein therapeutic”), and the peptide ligand will be an amino acid extension of the C- or N-terminus of the protein therapeutic. In addition, a short amino acid linker sequence may lie between the protein therapeutic and the peptide ligand. In this scenario, the peptide ligand, optional linker and protein therapeutic will be coded for by a nucleic acid comprising a sequence encoding protein therapeutic operably linked to (in the sense that the DNA sequences are contiguous and in reading frame) an optional linker sequence encoding a short polypeptide as described below, and a sequence encoding the peptide ligand. In this typical scenario, the peptide ligand is considered to be “conjugated” to the protein therapeutic optionally via a linker sequence. In a related embodiment, the peptide ligand amino acid sequence may interrupt or replace a section of the protein therapeutic amino acid sequence, provided, of course, that the insertion of the peptide ligand amino acid sequence does not interfere with the function of the protein therapeutic. In this embodiment, the “conjugate” may be coded for by a nucleic acid comprising a sequence encoding protein therapeutic interrupted by and operably linked to a sequence encoding the peptide ligand. In a further typical embodiment, the peptide will be linked, e.g. by chemical conjugation to the protein therapeutic or other therapeutic optionally via a linker sequence. Typically, according to this embodiment, the peptide ligand will be linked to the protein therapeutic via a side chain of an amino acid somewhere in the middle of the protein therapeutic that doesn't interfere with the therapeutic's activity. Here again, the peptide is considered to be “conjugated” to the therapeutic.

[0045] As used within the context of the present invention the term “target molecule” includes, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, and the like. Target molecules include, for example, extracellular molecules such as various serum factors including but not limited to plasma proteins such as serum albumin, immunoglobulins, apolipoproteins or transferrin, or proteins found on the surface of erythrocytes or lymphocytes, provided, of course, that binding of the peptide ligand to the cell surface protein does not substantially interfere with the normal function of the cell.

[0046] “Antibodies” and “immunoglobulins” are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains.

[0047] Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments or regions, each with a single antigen-binding site, and a residual “Fc” fragment or region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.

[0048] Pepsin treatment yields an F(ab′)₂ fragment that has two antigen-combining sites and is still capable of cross-linking antigen. The Fab′ fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.

[0049] “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

[0050] “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.

[0051] A “disorder” is any condition that would benefit from treatment with the compositions comprising the peptide ligands of the invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.

[0052] “Elimination half-time” is used in its ordinary sense, as is described in Goodman and Gillman's The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and Alfred Gilman, eds., 6th ed. 1980). Briefly, the term is meant to encompass a quantitative measure of the time course of drug elimination. The elimination of most drugs is exponential (i.e., follows first-order kinetics), since drug concentrations usually do not approach those required for saturation of the elimination process. The rate of an exponential process may be expressed by its rate constant, k, which expresses the fractional change per unit of time, or by its half-time, t_(1/2), the time required for 50% completion of the process. The units of these two constants are time⁻¹ and time, respectively. A first-order rate constant and the half-time of the reaction are simply related (k×t_(1/2)=0.693) and may be interchanged accordingly. Since first-order elimination kinetics dictates that a constant fraction of drug is lost per unit time, a plot of the log of drug concentration versus time is linear at all times following the initial distribution phase (i.e. after drug absorption and distribution are complete). The half-time for drug elimination can be accurately determined from such a graph.

[0053] “Transfection” refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaPO₄ precipitation and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell.

[0054] “Transformation” means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in section 1.82 of Sambrook et al., Molecular Cloning (2nd ed.), Cold Spring Harbor Laboratory, NY (1989), is generally used for prokaryotes or other cells that contain substantial cell-wall barriers. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., (1983) Gene, 23:315 and WO 89/05859 published 29 Jun. 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method described in sections 16.30-16.37 of Sambrook et al., supra, is preferred. General aspects of mammalian cell host system transformations have been described by Axel in U.S. Pat. No. 4,399,216 issued 16 Aug. 1983. Transformations into yeast are typically carried out according to the method of Van Solingen et al., (1977) J. Bact., 130:946 and Hsiao et al., (1979) Proc. Natl. Acad. Sci. (USA), 76:3829. However, other methods for introducing DNA into cells such as by nuclear injection, electroporation, or by protoplast fusion may also be used.

[0055] As used herein, the term “pulmonary administration” refers to administration of a formulation of the invention through the lungs by inhalation. As used herein, the term “inhalation” refers to intake of air to the alveoli. In specific examples, intake can occur by self-administration of a formulation of the invention while inhaling, or by administration via a respirator, e.g., to an patient on a respirator. The term “inhalation” used with respect to a formulation of the invention is synonymous with “pulmonary administration.”

[0056] As used herein, the term “parenteral” refers to introduction of a compound of the invention into the body by other than the intestines, and in particular, intravenous (i.v.), intraarterial (i.a.), intraperitoneal (i.p.), intramuscular (i.m.), intraventricular, and subcutaneous (s.c.) routes.

[0057] As used herein, the term “aerosol” refers to suspension in the air. In particular, aerosol refers to the particlization of a formulation of the invention and its suspension in the air. According to the present invention, an aerosol formulation is a formulation comprising a compound of the present invention that is suitable for aerosolization, i.e., particlization and suspension in the air, for inhalation or pulmonary administration.

[0058] II. Modes for Carrying Out the Invention

[0059] A. Peptide Ligands

[0060] Peptide ligands within the context of the present invention bind a target, preferably a serum protein such as serum albumin or an immunoglobulin, and can be identified in a direct binding assay, or by their ability to compete for target binding with a known ligand for the target. Preferred peptide ligands that bind serum albumin include linear and cyclic peptides, preferably cyclic peptide compounds comprising the following formulae or are peptides that compete for binding serum albumin of a particular mammalian species with peptides of the following formulae: (Xaa)_(x)-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-(Xaa)_(z) (Xaa)_(x)-Phe-Cys-Xaa-Asp-Trp-Pro-Xaa-Xaa-Xaa-Ser-Cys-(Xaa)_(z) (SEQ ID NO: 113) (Xaa)_(x)-Val-Cys-Tyr-Xaa-Xaa-Xaa-Ile-Cys-Phe-(Xaa)_(z) (SEQ ID NO: 114) (Xaa)_(x)-Cys-Tyr-Xaa₁-Pro-Gly-Xaa-Cys-(Xaa)_(z) (SEQ ID NO: 115) and (Xaa)_(x)-Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp-(Xaa)_(z) (SEQ ID NO: 116)

[0061] Preferred are peptide compounds of the foregoing general formulae wherein Xaa is an amino acid and x and z are a whole number greater or equal to 0 (zero), generally less than 100, preferably less than 10 and more preferably 0, 1, 2, 3, 4 or 5 and more preferably 4 or 5 and wherein Xaa₁ is selected from the group consisting of Ile, Phe, Tyr and Val.

[0062] Further preferred peptide ligands that bind a serum albumin are identified as described herein in the context of the following general formulae (Xaa)_(x)-Trp-Cys-Asp-Xaa-Xaa-Leu-Xaa-Ala-Xaa-Asp-Leu-Cys-(Xaa)_(z) (SEQ ID NO: 117) and (Xaa)_(x)-Asp-Leu-Val-Xaa-Leu-Gly-Leu-Glu-Cys-Trp-(Xaa)_(z) (SEQ ID NO: 118) wherein Xaa is an amino acid and x and z are a whole number greater or equal to zero, generally less than 100, preferably less than 10 and more preferably 0, 1, 2, 3, 4 or 5 and more preferably 4 or 5.

[0063] According to this aspect of the invention reference is made to the Figures and especially FIGS. 5A and 5B, 8A, 8B and 8C and FIG. 9 for exemplary peptides and appropriate amino acids for selecting peptides ligands that bind a mammalian serum albumin. In a preferred aspect, reference is made to FIG. 9 for selecting peptide ligands that bind accross several species of serum albumin.

[0064] Preferred compounds according to this aspect of the invention include: Asp-Leu-Cys-Leu-Arg-Asp-Trp-Gly-Cys-Leu-Trp (SEQ ID NO: 119) Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp (SEQ ID NO: 120) Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp (SEQ ID NO: 121) Gln-Arg-Leu-Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-Asp-Phe (SEQ ID NO: 122) Gln-Gly-Leu-Ile-Gly-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Gly-Asp-Ser-Val (SEQ ID NO: 123) Gln-Gly-Leu-Ile-Gly-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Gly-Asp-Ser-Val-Lys (SEQ ID NO: 124) Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-Asp (SEQ ID NO: 125) Arg-Leu-Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-Asp (SEQ ID NO: 126) Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-Asp (SEQ ID NO: 127) Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp (SEQ ID NO: 121) Arg-Leu-Met-Glu-Asp-Ile-Cys-Leu-Ala-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-Asp (SEQ ID NO: 128) Glu-Val-Arg-Ser-Phe-Cys-Thr-Asp-Trp-Pro-Ala-Glu-Lys-Ser-Cys-Lys-Pro-Leu-Arg-Gly (SEQ ID NO: 129) Arg-Ala-Pro-Glu-Ser-Phe-Val-Cys-Tyr-Trp-Glu-Thr-Ile-Cys-Phe-Glu-Arg-Ser-Glu-Gln (SEQ ID NO: 130) Glu-Met-Cys-Tyr-Phe-Pro-Gly-Ile-Cys-Trp-Met (SEQ ID NO: 131)

[0065] In a preferred embodiment, peptide ligands of the present invention bind IgG-Fc and can be identified by their ability to compete for binding of IgG-Fc in an in vitro assay with a peptide ligand having the general formula:

[0066] Xaa_(i)-Cys-Xaa_(j)-Cys-Xaa_(k), wherein Xaa_(i) is absent or is a peptide of between 1 and 4 amino acids, preferably 4 amino acids; X_(j) is preferably 9 amino acids having a preferred sequence Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Leu-Val-Trp (SEQ ID NO: 9); or Xaa-Xaa-Xaa-Xaa-Gly-Glu-Leu-Val-Trp (SEQ ID NO: 10); or Xaa₁-Xaa₂-Xaa₃-Xaa₄-Gly-Glu-Leu-Val-Trp (SEQ ID NO: 10), wherein Xaa₁ is Ala, Ser, or Thr; Xaa₂ is Trp or Tyr; Xaa₃ is His, or Trp; Xaa₄ is Leu or Met, and Xaa_(k) is absent or between 1 and 5 amino acids, preferably 5 amino acids, so long as the cyclic peptide or analog thereof retains the qualitative biological activity of binding IgG-Fc described above.

[0067] Preferred among this group of compounds are compounds comprising the sequence: Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Leu-Val-Trp-Cys-Xaa-Xaa-Xaa- (SEQ ID NO: 11) Xaa-Xaa; Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Gly-Glu-Leu-Val-Trp-Cys-Xaa-Xaa-Xaa- (SEQ ID NO: 12) Xaa-Xaa; Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Gly-Glu-Leu-Val-Trp-Cys-Xaa₉- (SEQ ID NO: 13) Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃, wherein Xaa₅ is Ala, Ser, or Thr; Xaa₆ is Trp or Tyr; Xaa₇ is His, or Trp; and Xaa₈ is Leu or Met; and Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Gly-Glu-Leu-Val-Trp-Cys-Xaa₉- (SEQ ID NO: 14) Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃ wherein Xaa₄ is Ser, Arg, or Asp; Xaa₅ is Ala, Ser, or Thr; Xaa₆ is Trp, Tyr; Xaa₇ is His, or Trp; Xaa₈ is Leu or Met; and Xaa₉ is Glu, Ser, Thr or Val.

[0068] In particular embodiments, the IgG-Fc binding peptide ligands of the present invention will compete with any of the peptide ligands represented in SEQ ID NO: 2-SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 11-SEQ ID NO: 111 described herein and preferably will compete with SEQ ID NO: 8 for binding IgG-Fc.

[0069] In another preferred embodiment, peptide ligands of the present invention bind human serum albumin and can be identified by their ability to compete for binding of human serum albumin in an in vitro assay with peptide ligands having the general formulae: (Xaa)_(x)-Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp-(Xaa)_(z) (SEQ ID NO: 116) (Xaa)_(x)-Phe-Cys-Xaa-Asp-Trp-Pro-Xaa-Xaa-Xaa-Ser-Cys-(Xaa)_(z) (SEQ ID NO: 113) (Xaa)_(x)-Val-Cys-Tyr-Xaa-Xaa-Xaa-Ile-Cys-Phe-(Xaa)_(z) (SEQ ID NO: 114) or (Xaa)_(x)-Cys-Tyr-Xaa₁-Pro-Gly-Xaa-Cys-(Xaa)₂ (SEQ ID NO: 115)

[0070] wherein Xaa is an amino acid, x and z are preferably 4 or 5 and Xaa₁ is selected from the group consisting of Ile, Phe, Tyr and Val.

[0071] In particular embodiments, the human serum albumin binding peptide ligands of the present invention will compete with any of the peptide ligands represented in SEQ ID NO: 120-131 described herein above and preferably will compete with SEQ ID NO: 122 for binding human serum albumin.

[0072] As will be appreciated from the foregoing, the term “compete” and “ability to compete” are relative terms. Thus the terms, when used to describe the peptide ligands of the present invention, refer to peptide ligands that produce a 50% inhibition of binding of, for example SEQ ID NO: 8 or SEQ ID NO: 122, when present at 50 μM, preferably when present at 1 μM, more preferably 100 nM, and preferably when present at 1 nM or less in a standard competition assay as described herein. Such peptide ligands generally will bind IgG-Fc with an affinity of less than 1 μM, preferably less than about 100 nM and more preferably less than about 10 nM as determined by a standard competition assay such as the one described in the Example sections. However, peptide ligands having an affinity for a serum protein such as serum albumin or IgG-Fc of less than about 1 nM and preferably between about 1 pM and 1 nM are equally likely to be peptide ligands within the context of the present invention.

[0073] For in vitro assay systems to determine whether a peptide or other compound has the “ability” to compete with a peptide ligand for binding to an IgG-Fc (or other plasma protein such as, e.g., serum albumin) as noted herein, the skilled artisan can employ any of a number of standard competition assays. Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of ligand. The amount of analyte in the test sample is inversely proportional to the amount of standard that becomes bound to the ligand.

[0074] Thus, the skilled artisan may determine whether a peptide or other compound has the ability to compete with a peptide ligand for binding to an IgG-Fc (or other target such as a plasma protein) employing procedures which include but are not limited to competitive assay systems using techniques such as radioimmunoassays (RIA), enzyme immunoassays (EIA), preferably the enzyme linked immunosorbent assay (ELISA), “sandwich” immunoassays, immunoradiometric assays, fluorescent immunoassays, and immunoelectrophoresis assays, to name but a few.

[0075] For these purposes the selected peptide ligand will be labeled with a detectable moiety (the detectably labeled peptide ligand hereafter called the “tracer”) and used in a competition assay with a candidate compound for binding IgG-Fc domain or other target. Numerous detectable labels are available which can be preferably grouped into the following categories:

[0076] (a) Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The peptide compound can be labeled with the radioisotope using the techniques described in Coligen et al., eds., Current Protocols in Immunology, Volumes 1 and 2 (1991), Wiley-Interscience, New York, N.Y., for example and radioactivity can be measured using scintillation counting.

[0077] (b) Fluorescent labels such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lissamine, phycoerythrin and Texas Red are available. The fluorescent labels can be conjugated to the peptide compounds using the techniques disclosed in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a fluorimeter.

[0078] (c) Various enzyme-substrate labels are available and U.S. Pat. No. 4,275,149 provides a review of some of these. The enzyme preferably catalyzes a chemical alteration of the chromogenic substrate which can be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light which can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehyrogenase, urease, peroxidase such as horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.

[0079] Examples of enzyme-substrate combinations include, for example:

[0080] (i) Horseradish peroxidase (BRP) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e.g. ABTS, orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidine hydrochloride (TMB));

[0081] (ii) alkaline phosphatase (AP) with para-nitrophenyl phosphate as chromogenic substrate; and

[0082] (iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g. p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase.

[0083] According to a particular assay, the tracer is incubated with immobilized target in the presence of varying concentrations of unlabeled candidate compound. Increasing concentrations of successful candidate compound effectively compete with binding of the tracer to immobilized target. The concentration of unlabeled candidate compound at which 50% of the maximally-bound tracer is displaced is referred to as the “IC₅₀” and reflects the IgG binding affinity of the candidate compound. Therefore a candidate compound with an IC₅₀ of 1 mM displays a substantially weaker interaction with the target than a candidate compound with an IC₅₀ of 1 μM.

[0084] In some phage display ELISA assays, binding affinity of a mutated (“mut”) sequence was directly compared of a control (“con”) peptide using methods described in B. C. Cunningham, D. G. Lowe, B. Li, B. D. Bennett, and J. A. Wells, EMBO J. 13:2508 (1994) and characterized by the parameter EC₅₀. Assays were performed under conditions where EC₅₀(con)/EC₅₀(mut) will approximate K_(d)(con)/K_(d)(mut).

[0085] Accordingly, the invention provides compounds “having the ability to compete” for target molecules such as IgG or human serum albumin binding in an in vitro assay as described. Preferably the compound has an IC₅₀ for the target such as IgG or human serum albumin of less than 1 μM. Preferred among these compound are compounds having an IC₅₀ of less than about 100 nM and preferably less than about 10 nM or less than about 1 nM. In further preferred embodiments according to this aspect of the invention the compounds display an IC₅₀ for the target molecule such as IgG or human serum albumin of less than about 100 pM and more preferably less than about 10 pM.

[0086] A preferred in vitro assay for the determination of a candidate compound's ability to compete with a peptide ligand described herein is as follows and is described more fully in the Examples. In preferred embodiments the candidate compound is a peptide. The ability of a candidate compound to compete with a labeled peptide ligand tracer for binding to IgG or human serum albumin is monitored using an ELISA. Dilutions of a candidate compound in buffer are added to microtiter plates coated with IgG or human serum albumin (as described in the Example Sections) along with tracer for 1 hr. The microtiter plate is washed with wash buffer and the amount of tracer bound to IgG or human serum albumin measured.

[0087] B. Peptide Ligand Combinations

[0088] According to the present invention, the peptide ligand is optionally linked to a bioactive compound to form a hybrid molecule that comprises a peptide ligand domain and an active domain. The bioactive compounds of the invention include any compound useful as a therapeutic or diagnostic agent. Non-limiting examples of bioactive compounds include polypeptides such as enzymes, hormones, cytokines, antibodies or antibody fragments, as well as organic compounds such as analgesics, antipyretics, antiinflammatory agents, antibiotics, antiviral agents, anti-fungal drugs, cardiovascular drugs, drugs that affect renal function and electrolyte metabolism, drugs that act on the central nervous system, chemotherapeutic drugs, etc. According to the present invention the peptide ligand domain is joined to an active domain, optionally via a flexible linker domain.

[0089] The hybrid molecules of the present invention are constructed by combining a peptide ligand domain with a suitable active domain. Depending on the type of linkage and its method of production, the peptide ligand domain may be joined via its N- or C-terminus to the N- or C-terminus of the active domain. For example, when preparing the hybrid molecules of the present invention via recombinant techniques, nucleic acid encoding a peptide ligand will be operably linked to nucleic acid encoding the active domain sequence, optionally via a linker domain. Typically the construct encodes a fusion protein wherein the C-terminus of the peptide ligand is joined to the N-terminus of the active domain. However, especially when synthetic techniques are employed, fusions where, for example, the N-terminus of the peptide ligand is joined to the N- or C-terminus of the active domain also are possible. In some instances, the peptide ligand domain may be inserted within the active domain molecule rather than being joined to the active domain at its N- or C-terminus. This configuration may be used to practice the invention so long as the functions of the peptide ligand domain and the active domain are preserved. For example, a peptide ligand may be inserted into a non-binding light chain CDR of an immunoglobulin without interfering with the ability of the immunoglobulin to bind to its target. Regions of active domain molecules that can accommodate peptide ligand domain insertions may be identified empirically (i.e. by selecting an insertion site, randomly, and assaying the resulting conjugate for the function of the active domain), or by sequence comparisons amongst a family of related active domain molecules (e.g., for active domains that are proteins) to locate regions of low sequence homology. Low sequence homology regions are more likely to tolerate insertions of peptide ligands domains than are regions that are well-conserved. For active domain molecules whose three-dimensional structures are known (e.g. from X-ray crystallographic or NMR studies), the three-dimensional structure may provide guidance as to peptide ligand insertion sites. For example, loops or regions with high mobility (i.e., large temperature or “B” factors) are more likely to accommodate peptide ligand domain insertions than are highly ordered regions of the structure, or regions involved in ligand binding or catalysis.

[0090] C. Linker Domains

[0091] According to the present invention, the peptide ligand domain is optionally linked to the active domain via a linker. The linker component of the hybrid molecule of the invention does not necessarily participate in but may contribute to the function of the hybrid molecule. Therefore, according to the present invention, the linker domain, is any group of molecules that provides a spatial bridge between the active domain and the peptide ligand domain.

[0092] The linker domain can be of variable length and makeup, however, according to the present invention, it is the length of the linker domain and not its structure that is important. The linker domain preferably allows for the peptide ligand domain of the hybrid molecule to bind, substantially free of steric and/or conformational restrictions to the target molecule. Therefore, the length of the linker domain is dependent upon the character of the two “functional” domains of the hybrid molecule, i.e., the peptide ligand domain and the active domain.

[0093] One skilled in the art will recognize that various combinations of atoms provide for variable length molecules based upon known distances between various bonds (Morrison, and Boyd, Organic Chemistry, 3rd Ed, Allyn and Bacon, Inc., Boston, Mass. (1977)). For example, the linker domain may be a polypeptide of variable length. The amino acid composition of the polypeptide determines the character and length of the linker. In a preferred embodiment, the linker molecule comprises a flexible, hydrophilic polypeptide chain. Exemplary, linker domains comprises one or more Gly and or Ser residues, such as those described in the Example sections herein.

[0094] D. Recombinant Synthesis

[0095] The present invention encompasses a composition of matter comprising an isolated nucleic acid, preferably DNA, encoding a peptide ligand or a hybrid molecule comprising a peptide ligand domain and a polypeptide active domain as described herein. DNAs encoding the peptides of the invention can be prepared by a variety of methods known in the art. These methods include, but are not limited to, chemical synthesis by any of the methods described in Engels et al. (1989), Agnew. Chem. Int. Ed. Engl. 28:716-734, the entire disclosure of which is incorporated herein by reference, such as the triester, phosphite, phosphoramidite and H-phosphonate methods. In one embodiment, codons preferred by the expression host cell are used in the design of the encoding DNA. Alternatively, DNA encoding the peptides of the invention can be altered to encode one or more variants by using recombinant DNA techniques, such as site specific mutagenesis (Kunkel et al. (1991), Methods Enzymol., 204:125-139; Carter et al. (1986), Nucl. Acids Res. 13:4331; Zoller et al. (1982), Nucl. Acids Res. 10:6487), cassette mutagenesis (Wells et al. (1985), Gene 34:315), restriction selection mutagenesis (Carter, Directed Mutagenesis: A Practical Approach (M. J. McPherson, ed.) IRL Press, Oxford, 1991), and the like.

[0096] According to preferred aspects described above, the nucleic acid encodes a peptide ligand capable of binding a target molecule. Target molecules include, for example, extracellular molecules such as various serum factors including but not limited to plasma proteins such as serum albumin, immunoglobulins, apolipoproteins or transferrin, or proteins found on the surface of erythrocytes or lymphocytes, provided, of course, that binding of the peptide ligand to the cell surface protein does not substantially interfere with the normal function of the cell.

[0097] According to another preferred aspect of the invention, the nucleic acid encodes a hybrid molecule comprising a peptide ligand domain sequence and an active domain. In this aspect of the invention, the active domain may comprise any polypeptide compound useful as a therapeutic or diagnostic agent, e.g., enzymes, hormones, cytokines, antibodies or antibody fragments. The nucleic acid molecule according to this aspect of the present invention encodes a hybrid molecule and the nucleic acid encoding the peptide ligand domain sequence is operably linked to (in the sense that the DNA sequences are contiguous and in reading frame) the nucleic acid encoding the biologically active agent. Optionally these DNA sequences may be linked through a nucleic acid sequence encoding a linker domain amino acid sequence.

[0098] According to this aspect, the invention further comprises an expression control sequence operably linked to the DNA molecule encoding a peptide of the invention, an expression vector, such as a plasmid, comprising the DNA molecule, wherein the control sequence is recognized by a host cell transformed with the vector, and a host cell transformed with the vector. In general, plasmid vectors contain replication and control sequences which are derived from species compatible with the host cell. The vector ordinarily carries a replication site, as well as sequences which encode proteins that are capable of providing phenotypic selection in transformed cells.

[0099] For expression in prokaryotic hosts, suitable vectors include pBR322 (ATCC No. 37,017), phGH107 (ATCC No. 40,011), pBO475, pS0132, pRIT5, any vector in the pRIT20 or pRMI30 series (Nilsson and Abrahmsen (1990), Meth. Enzymol. 185:144-161), pRIT2T, pKK233-2, pDR540 and pPL-lambda. Prokaryotic host cells containing the expression vectors of the present invention include E. coli K12 strain 294 (ATCC NO. 31,446), E. coli strain JM101 (Messing et al. (1981), Nucl. Acid Res. 9:309), E. coli strain B, E. coli strain_(—)1776 (ATCC No. 31537), E. coli c600, E. coli W3110 (F-, gamma-, prototrophic, ATCC No. 27,325), E. coli strain 27C7 (W3110, tonA, phoA E15, (argF-lac)169, ptr3, degP41, ompT, kan^(r)) (U.S. Pat. No. 5,288,931, ATCC No. 55,244), Bacillus subtilis, Salmonella typhimurium, Serratia marcesans, and Pseudomonas species.

[0100] In addition to prokaryotes, eukaryotic organisms, such as yeasts, or cells derived from multicellular organisms can be used as host cells. For expression in yeast host cells, such as common baker's yeast or Saccharomyces cerevisiae, suitable vectors include episomally-replicating vectors based on the 2-micron plasmid, integration vectors, and yeast artificial chromosome (YAC) vectors. For expression in insect host cells, such as Sf9 cells, suitable vectors include baculoviral vectors. For expression in plant host cells, particularly dicotyledonous plant hosts, such as tobacco, suitable expression vectors include vectors derived from the Ti plasmid of Agrobacterium tumefaciens.

[0101] Examples of useful mammalian host cells include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al. (1977), J. Gen Virol 36:59); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin (1980), Proc. Natl. Acad. Sci. USA 77:4216); mouse sertoli cells (TM4, Mather (1980), Biol. Reprod. 23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al. (1982), Annals N.Y. Acad. Sci. 383:44-68); MRC 5 cells; FS4 cells; and a human hepatoma cell line (Hep G2). For expression in mammalian host cells, useful vectors include vectors derived from SV40, vectors derived from cytomegalovirus such as the pRK vectors, including pRK5 and pRK7 (Suva et al. (1987), Science 237:893-896; EP 307,247 (Mar. 15, 1989), EP 278,776 (Aug. 17, 1988)) vectors derived from vaccinia viruses or other pox viruses, and retroviral vectors such as vectors derived from Moloney's murine leukemia virus (MoMLV).

[0102] Optionally, the DNA encoding the peptide of interest is operably linked to a secretory leader sequence resulting in secretion of the expression product by the host cell into the culture medium. Examples of secretory leader sequences include STII, ecotin, lamB, herpes GD, lpp, alkaline phosphatase, invertase, and alpha factor. Also suitable for use herein is the 36 amino acid leader sequence of protein A (Abrahmsen et al. (1985), EMBO J. 4:3901).

[0103] Host cells are transfected and preferably transformed with the above-described expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0104] Prokaryotic host cells used to produce the present peptides can be cultured as described generally in Sambrook et al., supra.

[0105] The mammalian host cells used to produce peptides of the invention can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM, Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham and Wallace (1979), Meth. in Enz. 58:44, Barnes and Sato (1980), Anal. Biochem. 102:255, U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195; U.S. Pat. No. Re. 30,985; or U.S. Pat. No. 5,122,469, the disclosures of all of which are incorporated herein by reference, may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as Gentamycin™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

[0106] The host cells referred to in this disclosure encompass cells in in vitro culture as well as cells that are within a host animal.

[0107] E. Chemical Synthesis

[0108] Another method of producing the compounds of the invention involves chemical synthesis. This can be accomplished by using methodologies well known in the art (see Kelley, R. F. & Winkler, M. E. in Genetic Engineering Principles and Methods, Setlow, J. K., ed., Plenum Press, N.Y., Vol. 12, pp 1-19 (1990); Stewart, J. M. Young, J. D., Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, Ill. (1984); see also U.S. Pat. Nos. 4,105,603; 3,972,859; 3,842,067; and 3,862,925).

[0109] Peptide ligands of the invention can be prepared conveniently using solid-phase peptide synthesis. Merrifield (1964), J. Am. Chem. Soc. 85:2149; Houghten (1985), Proc. Natl. Acad. Sci. USA 82:5132. Solid-phase peptide synthesis also can be used to prepare the hybrid molecule compositions of the invention if the active domain is or comprises a polypeptide.

[0110] Solid-phase synthesis begins at the carboxy terminus of the nascent peptide by coupling a protected amino acid to an inert solid support. The inert solid support can be any macromolecule capable of serving as an anchor for the C-terminus of the initial amino acid. Typically, the macromolecular support is a cross-linked polymeric resin (e.g., a polyamide or polystyrene resin) as shown in FIGS. 1-1 and 1-2, on pages 2 and 4 of Stewart and Young, supra. In one embodiment, the C-terminal amino acid is coupled to a polystyrene resin to form a benzyl ester. A macromolecular support is selected such that the peptide anchor link is stable under the conditions used to deprotect the α-amino group of the blocked amino acids in peptide synthesis. If a base-labile α-protecting group is used, then it is desirable to use an acid-labile link between the peptide and the solid support. For example, an acid-labile ether resin is effective for base-labile Fmoc-amino acid peptide synthesis as described on page 16 of Stewart and Young, Supra. Alternatively, a peptide anchor link and α-protecting group that are differentially labile to acidolysis can be used. For example, an aminomethyl resin such as the phenylacetamidomethyl (Pam) resin works well in conjunction with Boc-amino acid peptide synthesis as described on pages 11-12 of Stewart and Young, supra.

[0111] After the initial amino acid is coupled to an inert solid support, the α-amino protecting group of the initial amino acid is removed with, for example, triluoroacetic acid (TFA) in methylene chloride and neutralized in, for example, triethylamine (TEA). Following deprotection of the initial amino acid's amino group, the next α-amino and side chain protected amino acid in the synthesis is added. The remaining α-amino and, if necessary, side chain protected amino acids are then coupled sequentially in the desired order by condensation to obtain an intermediate compound connected to the solid support. Alternatively, some amino acids may be coupled to one another to form a fragment of the desired peptide followed by addition of the peptide fragment to the growing solid phase peptide chain.

[0112] The condensation reaction between two amino acids, or an amino acid and a peptide, or a peptide and a peptide can be carried out according to the usual condensation methods such as the axide method, mixed acid anhydride method, DCC(N,N′-dicyclohexylcarbodiimide) or DIC (N,N′-diisopropylcarbodiimide) methods, active ester method, p-nitrophenyl ester method, BOP (benzotriazole-1-yl-oxy-tris [dimethylamino] phosphonium hexafluorophosphate) method, N-hydroxysuccinic acid imido ester method, etc., and Woodward reagent K method.

[0113] It is common in the chemical synthesis of peptides to protect any reactive side chain groups of the amino acids with suitable protecting groups. Ultimately, these protecting groups are removed after the desired polypeptide chain has been sequentially assembled. Also common is the protection of the α-amino group on an amino acid or peptide fragment while the C-terminal carboxy group of the amino acid or peptide fragment reacts with the free N-terminal amino group of the growing solid phase polypeptide chain, followed by the selective removal of the α-amino group to permit the addition of the next amino acid or peptide fragment to the solid phase polypeptide chain. Accordingly, it is common in polypeptide synthesis that an intermediate compound is produced which contains each of the amino acid residues located in the desired sequence in the peptide chain wherein individual residues still carry side-chain protecting groups. These protecting groups can be removed substantially at the same time to produce the desired polypeptide product following removal from the solid phase.

[0114] α- and ε-amino side chains can be protected with benzyloxycarbonyl (abbreviated Z), isonicotinyloxycarbonyl (iNOC), o-chlorobenzyloxycarbonyl [Z(2Cl)], p-nitrobenzyloxycarbonyl [ZNO₂)], p-methoxybenzyloxycarbonyl [Z(OMe)], t-butoxycarbonyl (Boc), t-amyloxycarbonyl (Aoc), isobornyloxycarbonyl adamantyloxycarbonyl, 2-(4-biphenyl)-2-propyloxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl (Fmoc), methylsulfonyethoxycarbonyl (Msc), trifluoroacetyl phthalyl, formyl 2-nitrophenylsulphenyl (NPS), diphenylphosphinothioyl (Ppt), and dimethylphosphinothioyl (Mpt) groups, and the like.

[0115] Protective groups for the carboxy functional group are exemplified by benzyl ester (OBzl), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb), t-butyl ester (Obut), 4-pyridylmethyl ester (OPic), and the like. It is often desirable that specific amino acids such as arginine, cysteine, and serine possessing a functional group other than amino and carboxyl groups are protected by a suitable protective group. For example, the guanidino group of arginine may be protected with nitro, p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzesulfonyl, 4-methoxy-2,6-dimethylbenzenesulfonyl (Nds), 1,3,5-trimethylphenysulfonyl (Mts), and the like. The thiol group of cysteine can be protected with p-methoxybenzyl, trityl, and the like.

[0116] Many of the blocked amino acids described above can be obtained from commercial sources such as Novabiochem (San Diego, Calif.), Bachem CA (Torrence, Calif.) or Peninsula Labs (Belmont, Calif.).

[0117] Stewart and Young, supra, provides detailed information regarding procedures for preparing peptides. Protection of α-amino groups is described on pages 14-18, and side chain blockage is described on pages 18-28. A table of protecting groups for amine, hydroxyl and sulfhydryl functions is provided on pages 149-151.

[0118] After the desired amino acid sequence has been completed, the peptide can be cleaved away from the solid support, recovered and purified. The peptide is removed from the solid support by a reagent capable of disrupting the peptide-solid phase link, and optionally deprotects blocked side chain functional groups on the peptide. In one embodiment, the peptide is cleaved away from the solid phase by acidolysis with liquid hydrofluoric acid BF), which also removes any remaining side chain protective groups. Preferably, in order to avoid alkylation of residues in the peptide (for example, alkylation of methionine, cysteine, and tyrosine residues), the acidolysis reaction mixture contains thio-cresol and cresol scavengers. Following HF cleavage, the resin is washed with ether, and the free peptide is extracted from the solid phase with sequential washes of acetic acid solutions. The combined washes are lyophilized, and the peptide is purified.

[0119] F. Chemical Conjugation of Hybrids

[0120] In certain embodiments of the present invention, the hybrid molecules may comprise active domains that are organic compounds having diagnostic or therapeutic utility, or alternatively, fusions between a peptide ligand domain and a polypeptide active domain in configurations that cannot be encoded in a single nucleic acid. Examples of the latter embodiment include fusions between the amino terminus of a peptide ligand and the amino terminus of the active domain, or fusions between the carboxy-terminus of a peptide ligand and the carboxy-terminus of the active domain.

[0121] Chemical conjugation may be employed to prepare these embodiments of the hybrid molecule, using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene, 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0122] G. Disulfide-Linked Peptides

[0123] As described above, some embodiments of the invention include cyclized peptide ligands. Peptide ligands may be cyclized by formation of a disulfide bond between cysteine residues. Such peptides can be made by chemical synthesis as described above and then cyclized by any convenient method used in the formation of disulfide linkages. For example, peptides can be recovered from solid phase synthesis with sulfhydryls in reduced form, dissolved in a dilute solution wherein the intramolecular cysteine concentration exceeds the intermolecular cysteine concentration in order to optimize intramolecular disulfide bond formation, such as a peptide concentration of 25 mM to 1 μM, and preferably 500 μM to 1 μM, and more preferably 25 μM to 1 μM, and then oxidized by exposing the free sulfhydryl groups to a mild oxidizing agent that is sufficient to generate intramolecular disulfide bonds, e.g., molecular oxygen with or without catalysts such as metal cations, potassium ferricyanide, sodium tetrathionate, etc. Alternatively, the peptides can be cyclized as described in Pelton et al. (1986), J. Med. Chem. 29:2370-2375.

[0124] Cyclization can be achieved by the formation, for example, of a disulfide bond or a lactam bond between a first Cys and a second Cys. Residues capable of forming a disulfide bond include, for example, Cys, Pen, Mpr, and Mpp and its 2-amino group-containing equivalents. Residues capable of forming a lactam bridge include, for example, Asp Glu, Lys, Orn, αβ-diaminobutyric acid, diaminoacetic acid, aminobenzoic acid and mercaptobenzoic acid. The compounds herein can be cyclized for example via a lactam bond which can utilize the side chain group of a non-adjacent residue to form a covalent attachment to the N-terminus amino group of Cys or other amino acid. Alternative bridge structures also can be used to cyclize the compounds of the invention, including for example, peptides and peptidomimetics, which can cyclize via S—S, CH2—S, CH2—O—CH2, lactam ester or other linkages.

[0125] H. Pharmaceutical Compositions

[0126] Pharmaceutical compositions which comprise the hybrid molecules of the invention may be administered in any suitable manner, including parental, topical, oral, or local (such as aerosol or transdermal) or any combination thereof.

[0127] Other suitable compositions of the present invention comprise any of the above-noted compositions with a pharmaceutically acceptable carrier, the nature of the carrier differing with the mode of administration, for example, in oral administration, usually using a solid carrier and in i.v. administration, a liquid salt solution carrier.

[0128] The compositions of the present invention include pharmaceutically acceptable components that are compatible with the subject and the protein of the invention. These generally include suspensions, solutions and elixirs, and most especially biological buffers, such as phosphate buffered saline, saline, Dulbecco's Media, and the like. Aerosols may also be used, or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like (in the case of oral solid preparations, such as powders, capsules, and tablets).

[0129] As used herein, the term “pharmaceutically acceptable” generally means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0130] The formulation of choice can be accomplished using a variety of the aforementioned buffers, or even excipients including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin cellulose, magnesium carbonate, and the like. “PEGylation” of the compositions may be achieved using techniques known to the art (see for example International Patent Publication No. WO92/16555, U.S. Pat. No. 5,122,614 to Enzon, and International Patent Publication No. WO92/00748).

[0131] A preferred route of administration of the present invention is in the aerosol or inhaled form. The compounds of the present invention, combined with a dispersing agent, or dispersant, can be administered in an aerosol formulation as a dry powder or in a solution or suspension with a diluent.

[0132] As used herein, the term “dispersant” refers to a agent that assists aerosolization of the compound or absorption of the protein in lung tissue, or both. Preferably the dispersant is pharmaceutically acceptable. Suitable dispersing agents are well known in the art, and include but are not limited to surfactants and the like. For example, surfactants that are generally used in the art to reduce surface induced aggregation of a compound, especially a peptide compound, caused by atomization of the solution forming the liquid aerosol, may be used. Nonlimiting examples of such surfactants are surfactants such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitan fatty acid esters. Amounts of surfactants used will vary, being generally within the range of from about 0.001% to about 4% by weight of the formulation. In a specific aspect, the surfactant is polyoxyethylene sorbitan monooleate or sorbitan trioleate. Suitable surfactants are well known in the art, and can be selected on the basis of desired properties, depending on the specific formulation, concentration of the compound, diluent (in a liquid formulation) or form of powder (in a dry powder formulation), etc.

[0133] Moreover, depending on the choice of the peptide ligand, the desired therapeutic effect, the quality of the lung tissue (e.g., diseased or healthy lungs), and numerous other factors, the liquid or dry formulations can comprise additional components, as discussed further below.

[0134] The liquid aerosol formulations generally contain the peptide ligand/active domain hybrid and a dispersing agent in a physiologically acceptable diluent. The dry powder aerosol formulations of the present invention consist of a finely divided solid form of the peptide ligand/active domain hybrid and a dispersing agent. With either the liquid or dry powder aerosol formulation, the formulation must be aerosolized. That is, it must be broken down into liquid or solid particles in order to ensure that the aerosolized dose actually reaches the alveoli. In general the mass median dynamic diameter will be 5 micrometers or less in order to ensure that the drug particles reach the lung alveoli (Wearley, L. L. (1991), Crit. Rev. in Ther. Drug Carrier Systems 8:333). The term “aerosol particle” is used herein to describe the liquid or solid particle suitable for pulmonary administration, i.e., that will reach the alveoli. Other considerations such as construction of the delivery device, additional components in the formulation and particle characteristics are important. These aspects of pulmonary administration of a drug are well known in the art, and manipulation of formulations, aerosolization means and construction of a delivery device require at most routine experimentation by one of ordinary skill in the art.

[0135] With regard to construction of the delivery device, any form of aerosolization known in the art, including but not limited to nebulization, atomization or pump aerosolization of a liquid formulation, and aerosolization of a dry powder formulation, can be used in the practice of the invention. A delivery device that is uniquely designed for administration of solid formulations is envisioned. Often, the aerosolization of a liquid or a dry powder formulation will require a propellant. The propellant may be any propellant generally used in the art. Specific nonlimiting examples of such useful propellants are a chloroflourocarbon, a hydrofluorocarbon, a hydochlorofluorocarbon, or a hydrocarbon, including triflouromethane, dichlorodiflouromethane, dichlorotetrafuoroethanol, and 1,1,1,2-tetraflouroethane, or combinations thereof.

[0136] In a preferred aspect of the invention, the device for aerosolization is a metered dose inhaler. A metered dose inhaler provides a specific dosage when administered, rather than a variable dose depending on administration. Such a metered dose inhaler can be used with either a liquid or a dry powder aerosol formulation. Metered dose inhalers are well known in the art.

[0137] Once the peptide ligand/active domain hybrid reaches the lung, a number of formulation-dependent factors affect the drug absorption. It will be appreciated that in treating a disease or disorder that requires circulatory levels of the compound, such factors as aerosol particle size, aerosol particle shape, the presence or absence of infection, lung disease or emboli may affect the absorption of the compounds. For each of the formulations described herein, certain lubricators, absorption enhancers, protein stabilizers or suspending agents may be appropriate. The choice of these additional agents will vary depending on the goal. It will be appreciated that in instances where local delivery of the compounds is desired or sought, such variables as absorption enhancement will be less critical.

[0138] I. Liquid Aerosol Formulations

[0139] The liquid aerosol formulations of the present invention will typically be used with a nebulizer. The nebulizer can be either compressed air driven or ultrasonic. Any nebulizer known in the art can be used in conjunction with the present invention such as but not limited to: Ultravent, Mallinckrodt, Inc. (St. Louis, Mo.); the Acorn II nebulizer (Marquest Medical Products, Englewood Colo.). Other nebulizers useful in conjunction with the present invention are described in U.S. Pat. No. 4,624,251 issued Nov. 25, 1986; U.S. Pat. No. 3,703,173 issued Nov. 21, 1972; U.S. Pat. No. 3,561,444 issued Feb. 9, 1971 and U.S. Pat. No. 4,635,627 issued Jan. 13, 1971.

[0140] The formulation may include a carrier. The carrier is a macromolecule which is soluble in the circulatory system and which is physiologically acceptable where physiological acceptance means that those of skill in the art would accept injection of said carrier into a patient as part of a therapeutic regime. The carrier preferably is relatively stable in the circulatory system with an acceptable elimination half-time. Such macromolecules include but are not limited to soya lecithin, oleic acid and sorbetan trioleate, with sorbitan trioleate preferred.

[0141] The formulations of the present embodiment may also include other agents useful for protein stabilization or for the regulation of osmotic pressure. Examples of the agents include but are not limited to salts, such as sodium chloride, or potassium chloride, and carbohydrates, such as glucose, galactose or mannose, and the like.

[0142] J. Aerosol Dry Powder Formulations

[0143] It is also contemplated that the present pharmaceutical formulation will be used as a dry powder inhaler formulation comprising a finely divided powder form of the peptide ligand and a dispersant. The form of the compound will generally be a lyophilized powder. Lyophilized forms of peptide ligand/active domain hybrid compounds can be obtained through standard techniques.

[0144] In another embodiment, the dry powder formulation will comprise a finely divided dry powder containing one or more compounds of the present invention, a dispersing agent and also a bulking agent. Bulking agents useful in conjunction with the present formulation include such agents as lactose, sorbitol, sucrose, or mannitol, in amounts that facilitate the dispersal of the powder from the device.

[0145] K. Research, Manufacturing, and Diagnostic Compositions

[0146] In a preferred embodiment, the peptide ligands or the hybrid molecules of the invention are non-covalently adsorbed or covalently bound to a macromolecule, such as a solid support. It will be appreciated that the invention encompasses macromolecules complexed with the peptide ligands or hybrid molecules. In a preferred embodiment, the peptide ligands of the invention are directed against an immunoglobulin, such as, e.g., the IgG-Fc peptide ligands disclosed infra. Such peptide ligands may be used as affinity purification agents. In this process, the peptide ligands are immobilized on a solid phase support such as a Sephadex resin or filter paper, using methods well known in the art. The immobilized peptide ligand is contacted with a sample containing the immunoglobulin protein (or fragment thereof) to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the immunoglobulin protein, which is bound to the immobilized peptide ligand. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0, that will release the immunoglobulin protein from the peptide ligand.

[0147] In general, the solid support is an inert matrix, such as a polymeric gel, comprising a three-dimensional structure, lattice or network of a material. Almost any macromolecule, synthetic or natural, can form a gel in a suitable liquid when suitably cross-linked with a bifunctional reagent. Preferably, the macromolecule selected is convenient for use in affinity chromatography. Most chromatographic matrices used for affinity chromatography are xerogels. Such gels shrink on drying to a compact solid comprising only the gel matrix. When the dried xerogel is resuspended in the liquid, the gel matrix imbibes liquid, swells and returns to the gel state. Xerogels suitable for use herein include polymeric gels, such as cellulose, cross-linked dextrans (e.g. Sepharose), agarose, cross-linked agarose, polyacrylamide gels, and polyacrylamide-agarose gels.

[0148] Alternatively, aerogels can be used for affinity chromatography. These gels do not shrink on drying but merely allow penetration of the surrounding air. When the dry gel is exposed to liquid, the latter displaces the air in the gel. Aerogels suitable for use herein include porous glass and ceramic gels.

[0149] Also encompassed herein are the peptide ligands or hybrid molecules of the invention coupled to derivatized gels wherein the derivative moieties facilitate the coupling of the hybrid molecules to the gel matrix and avoid steric hindrance of the peptide ligand-target molecule interaction in affinity chromatography. Alternatively, spacer arms can be interposed between the gel matrix and the hybrid molecules for similar benefits.

[0150] A variation on the above contemplates the use of gene fusions and the use of the peptide ligands as purification reagents. According to this aspect of the invention the gene encoding a peptide ligand is associated, in a vector, with a gene encoding another protein or a fragment of another protein. This results in the peptide ligand being produced by the host cell as a fusion with another protein or peptide. The “other” protein or peptide is often a protein or peptide which can be secreted by the cell, making it possible to isolate and purify the other protein from the culture medium and eliminating the necessity of destroying the host cells which arises when the other protein remains inside the cell. Alternatively, the fusion protein can be expressed intracellularly. It is useful to use fusion proteins that are highly expressed.

[0151] The use of gene fusions is analogous to the use of Protein A fusions which are often used because the binding of protein A, or more specifically the Z domain of protein A binds to IgG and provides an “affinity handle” for the purification of the fused protein. According to a preferred aspect of the invention, peptide ligands which bind serum albumin are use as “affinity handles” for the purification of fused proteins on a solid serum albumin support. For example, a DNA sequence encoding the desired peptide ligand can be fused by site directed mutagenesis to the gene for protein. After expression and secretion, the fusion protein can be purified on a matix of serum albumin to which the peptide ligand will bind. After purification the peptide ligand can be enzymatically or chemically cleaved to yield free protein or left intact to aid in increasing the elimination half life of the fused protein. Fusion proteins can be cleaved using chemicals, such as cyanogen bromide, which cleaves at a methionine, or hydroxylamine, which cleaves between an Asn and Gly residue. Using standard recombinant DNA methodology, the nucleotide base pairs encoding these amino acids may be inserted just prior to the 5′ end of the gene encoding the desired peptide. Alternatively, one can employ proteolytic cleavage of fusion protein. Carter, in Protein Purification: From Molecular Mechanisms to Large-Scale Processes, Ladisch et al., eds. (American Chemical Society Symposium Series No. 427, 1990), Ch 13, pages 181-193.

[0152] The following examples are offered by way of illustration and not by way of limitation. The disclosures of all citations in the specification are expressly incorporated herein by reference.

EXAMPLE 1

[0153] IgG-Fc Peptide Ligands

[0154] An in vitro selection designed to identify peptide ligands which bind the IgG-Fc surface without the constraint that the peptides function in vivo was performed. The selection was accomplished using a combination of polyvalent and monovalent phage display which has recently been applied to generate peptides that bind a variety of cellular hormones and receptors. N. C. Wrighton, et al. (1996), Science 273:458, O. Livnah, et al. (1996), Science 273:464. A single disulfide-constrained peptide library was constructed that consisted of 4×10⁹ different peptides of the form Xaa_(i)-Cys-Xaa_(j)-Cys-Xaa_(k) wherein Xaa is a random amino acid from an NNS codon, i+j+k=18, and j=4 through 10. This library was expressed on the surface of M13 bacteriophage as an N-terminal fusion to the gene VIII protein with a short linker consisting of glycine and serine residues. H. B. Lowman et al. (1998), Biochemistry 37: 8870-8878. More particularly, the library construct contained an STII secretion signal peptide, the peptide library of twenty amino acid length, i.e., Xaa_(i)-Cys-Xaa_(j)-Cys-Xaa_(k) wherein Xaa is a random amino acid from an NNS codon, i+j+k=18, and j=4 through 10, a Gly-Gly-Gly-Ser-Gly-Gly-Gly linker (SEQ ID NO: 1), and the M13 gene VIII starting at the first residue of the mature protein.

[0155] In principle, peptides could be selected that bind to potentially any region of the IgG-Fc due to the unbiased nature of this library. However, after several rounds of selection, the library became dominated by a single peptide, Fc-I (Glu-Thr-Gln-Arg-Cys-Thr-Trp-His-Met-Gly-Glu-Leu-Val-Trp-Cys-Glu-Arg-Glu-His-Asn) (SEQ ID NO: 2). Selections were performed as described in H. B. Lowman, et al., supra, with the following modifications: microtiter wells were coated using 5 μg/ml IgG-Fc; Casein Blocker Buffer (Pierce) was used in place of 0.1% BSA to better prevent non-specific binding; elution of phage was effected with either 75 mM DTT or 0.2 mM glycine pH 2.0 with equivalent results. IgG-Fc was obtained by papain cleavage of CD4-IgG₁ immunoadhesin protein, Capon et al. (1989), Nature, 337: 525. Cleaved material was purified over Protein A Sepharose followed by Superdex-75 (Pharmacia) and then quantified by absorbance at 280 nm.

[0156] Repetition of the selection experiment again gave Fc-I and also a related peptide, Fc-II (Lys-Glu-Ala-Ser-Cys-Ser-Tyr-Trp-Leu-Gly-Glu-Leu-Val-Trp-Cys-Val-Ala-Gly-Val-Glu) (SEQ ID NO: 3). The Fc-II peptide shared the cysteine spacing and the internal Gly-Glu-Leu-Val-Trp (SEQ ID NO: 132) sequence seen in Fc-I. Apparently, these two peptides bound IgG-Fc with an affinity high enough to be selected over any of the other IgG-Fc binding peptides present in the starting pool. Both peptides were synthesized on solid phase using standard 9-fluorenylmethoxycarbonyl protocols and purified by reversed-phase HPLC. Masses were confirmed by electrospray mass spectrometry, and purified peptides were quantified by UV absorbance at 280 nm.

[0157] Competition ELISAs were performed in a manner similar to the method described in H. B. Lowman, et al., supra. Briefly, Protein A Z-domain was immobilized on microtiter wells at a concentration of 5 μg/ml, blocked, and washed as described. A matrix of mixtures of biotinylated-IgG-Fc at concentrations from 312 nM to 0.3 nM and peptide at concentrations from 215 μM to 0.8 nM was prepared. These mixtures were incubated with immobilized Protein A Z-domain for 1 hour. Plates were then washed and developed as described using avidin/HRP conjugate. Inhibition curves were then computed for each concentration of biotin-IgG-Fc, and then the curve of half-maximal inhibition, “IC₅₀”, was extrapolated to zero biotin-IgG-Fc concentration in order to obtain a K_(i). The Fc-I and Fc-II peptides both were found to compete with Protein A (Z-domain) (B. Nilsson et al. (1987), Protein Eng. 1:107) for binding to IgG-Fc with inhibition constants (K_(i)) of about 5 μM. The results imply that these peptides bind to an overlapping site on IgG-Fc that coincides with the Protein A binding site.

[0158] The DNA sequence of the Fc-II peptide was moved to a monovalent phage display format by cassette mutagenesis to give a construct with the STII signal sequence, the Fc-II peptide Lys-Glu-Ala-Ser-Cys-Ser-Tyr-Trp-Leu-Gly-Glu-Leu-Val-Trp-Cys-Val-Ala-Gly-Val-Glu (SEQ ID NO: 3), a Gly-Gly-Gly-Pro-Gly-Gly-Gly linker (SEQ ID NO: 4), and the M13 gene III protein starting at residue 253. The Fc-II sequence was affinity-matured by monovalent phage display. Five residue blocks were randomly mutated in six separate libraries to exhaustively cover the non-cysteine positions in the peptide sequence and then screened against IgG-Fc.

[0159] A series of second generation monovalent phage display libraries were constructed based on the Fc-II sequence Lys-Glu-Ala-Ser-Cys-Ser-Tyr-Trp-Leu-Gly-Glu-Leu-Val-Trp-Cys-Val-Ala-Gly-Val-Glu (SEQ ID NO: 3) in which five sequential residues were randomized using NNS codons in each library starting at positions 1, 4, 7, 10, 12, and 16, excluding the two cysteines. Each library had a diversity of approximately 1×10⁸. These libraries were independently screened for binding to IgG-Fc for six rounds and then sequenced. Preferred residues from this selection were then recombined using three additional libraries that spanned the entire peptide sequence. The three additional libraries were constructed using the degeneracy of the genetic code to recombine the preferred amino acids at each position into one peptide. The DNA sequences for these libraries contained the following mixtures of bases (IUPAC codes): DRG GWA GMA RRC TGC KCT TRS CAC MTG GGC GAG CTG GTC TGG TGC RVC RVM BKC GAS KDW (SEQ ID NO: 5), DRS VWG SVG RRC TGC KCC TRS YRS MTG GGC GAG CTG GTC TGG TGC RNC VVS NBS GWS KDM (SEQ ID NO: 6), and DNS NNS NNS VNS TGC BVG TDS HRS MDS GGC GAG STC KKG WRG TGC RNM NNS NNS NNS NNM (SEQ ID NO: 7). These libraries also were sorted against IgG-Fc for six rounds and then sequenced.

[0160] After screening against IgG-Fc, the consensus patterns from these libraries suggested a highly conserved 13-residue core sequence (Asp-Cys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr) (SEQ ID NO: 8). The corresponding peptide (Fc-III) was synthesized and found to inhibit binding of Protein A (Z-domain) to Fc with an IC₅₀ of 100 nM. Thus, although Fc-III is seven residues shorter than Fc-II, it binds 50-times more tightly. Despite its smaller size, the binding affinity of Fc-III to Fc was only ten-fold weaker than that of the domains from Protein A and Protein G, which are each about four times larger and bind with K_(d)S around 10 nM. S. R. Fahnestock, et al. in Bacterial Immunoglobulin-Binding Proteins (Academic Press, Inc. 1990) Vol. 1, chap. 11. R. Karlsson, L. Jendeberg, B. Nilsson, J. Nilsson, P. Nygren (1995), J. Immuno. Methods 183:43.

[0161] Table 1 lists the amino acid sequences and IgG-Fc binding affinities of exemplary IgG-Fc peptide ligands that were identified using the procedures described above. TABLE I IgG-Fc Peptide Ligand Sequences and Affinities Sequence Sequence ID NO Binding Affinity Peptides *All peptides have an N-terminal amine and a C-terminal amide KEASCSYWLGELVWCVAGVE SEQ ID NO: 3 5000 nM (K_(i)) ETQRCTWHMGELVWCEREHN SEQ ID NO: 2 5000 nM (K_(i)) DLADCSWHMGELVWCSRVEG SEQ ID NO: 15  50 nM (K_(d)) WEADCAWHLGELVWCTPMEF SEQ ID NO: 16  30 nM (IC₅₀)    DCAWHLGELVWCT SEQ ID NO: 8  100 nM (IC₅₀) Phage Clones (M13/gIII Display) All phage affinities are EC_(50s) N/A = Not individually assayed. Since they were selected for binding, EC₅₀ likely to be <1 uM or better. All of the peptides listed bind IgG-Fc. Focused Libraries KEASCSYWLGELVWCDTLTE SEQ ID NO: 17 N/A KEASCSYWLGELVWCSPGVE SEQ ID NO: 18  734 nM KEASCSYWLGELVWCSGVEG SEQ ID NO: 19 N/A KEASCSYWLGELVWCSAGVE SEQ ID NO: 20 N/A ESEDCSYWLGELVWCVAGVE SEQ ID NO: 21 N/A EKEDCSYWLGELVWCVAGVE SEQ ID NO: 22 N/A EDPDCSYWLGELVWCVAGVE SEQ ID NO: 23 N/A EEADCSYWLGELVWCVAGVE SEQ ID NO: 24 N/A NADDCSYWLGELVWCVAGVE SEQ ID NO: 25 N/A SETTCSYWLGELVWCVAGVE SEQ ID NO: 26 N/A AWKTCQWLGELVWCVAGVE SEQ ID NO: 27 N/A DLADCSYWLGELVWCSRVEG SEQ ID NO: 28  776 nM KEADCAWHLGELVWCVAGVE SEQ ID NO: 29  138 nM KEAECSYHLGELVWCVAGVE SEQ ID NO: 30 N/A KEARCWYWHGELVWCSDPEE SEQ ID NO: 31  809 nM KEASCSYHLGELVWCVAGVE SEQ ID NO: 32  416 nM KEASCSWHLGELVWCVAGVE SEQ ID NO: 33  225 nM KEASCSYWLGELVWCTEGVE SEQ ID NO: 34  818 nM KEASCSYWLGELVWCDDGVE SEQ ID NO: 35 N/A KEASCSYWLGELVWCSEGVE SEQ ID NO: 36 N/A KEASCSYWLGELVWCSPGVE SEQ ID NO: 18 N/A KEASCSYWLGEVWKCKSGVE SEQ ID NO: 37 N/A KEASCSYWLGELVWCDNGVE SEQ ID NO: 38 N/A KEASCSYWLGELVWCDTFDE SEQ ID NO: 39  301 nM KEASCSYWLGELVWCDGLDE SEQ ID NO: 40  326 nM KEASCSYWLGELVWCVGLDE SEQ ID NO: 41  278 nM KEASCSYWLGELVWCEDTLE SEQ ID NO: 42 N/A KEASCSYWLGELVWCEDTME SEQ ID NO: 43 N/A KEASCSYWLGELVWCEDMME SEQ ID NO: 44 N/A WVEDCSWHMGELVWCDGGEF SEQ ID NO: 45  139 nM KEASCSYWLGELVWCDWMNG SEQ ID NO: 46 N/A KEASCSYWLGELVWCDDTPV SEQ ID NO: 47 N/A KEASCSYWLGELVWCDDYGE SEQ ID NO: 48 N/A KEASCSYWLGELVWCSDLWE SEQ ID NO: 49 N/A WRGGCSWHMGELVWCEHDME SEQ ID NO: 50 N/A AVSKCSFHMGELVWCSDVMN SEQ ID NO: 51 N/A NQVSCSYSRGELVWCSKQSQ SEQ ID NO: 52 N/A GRMECAWHQGELVWCTPTLE SEQ ID NO: 53 N/A GTMECSWHQGELVWCTPTLA SEQ ID NO: 54 N/A EMRDCSWHLGELVWCAHMEG SEQ ID NO: 55 N/A GSWECAYHLGELVWCETGSG SEQ ID NO: 56 N/A VAEPCAYHLGELVWCEVLKG SEQ ID NO: 57 N/A KEAMCSYWLGELVWCESDMP SEQ ID NO: 58 N/A Designed Clones DLADCSWHLGELVWCSRVEG SEQ ID NO: 59   9 nM DLADCSWHLGELVWCVGLDE SEQ ID NO: 60  28 nM WVEDCSWHLGELVWCVGLDF SEQ ID NO: 61  31 nM Secondary Optimization KVADCAWHMGELVWCTEVEG SEQ ID NO: 62  23 nM GEEDCSYHLGELVMCTELDD SEQ ID NO: 63  69 nM GVADCAWHLGELVWCTERED SEQ ID NO: 64 N/A GEEDCAWHLGELVWCSGGDF SEQ ID NO: 65  100 nM WEADCAWHLGELVWCTKVEE SEQ ID NO: 66   7 nM GEADCSYHLGELVWCNDFEE SEQ ID NO: 67  156 nM WVDCAYHLGELVWCSTFEE SEQ ID NO: 68   9 nM WVEDCAWHMGELVWCTKVDE SEQ ID NO: 69  70 nM READCAWHLGELVWCSERDL SEQ ID NO: 70  47 nM EEASCAYHLGELVWCDAFDV SEQ ID NO: 71  77 nM RVASCAWHLGELVWCDGLDG SEQ ID NO: 72 N/A GEADCAWHLGELVWCTKVEE SEQ ID NO: 73  38 nM GEASCAYHLGELVWCDEGEG SEQ ID NO: 74  386 nM RVEDCAYHLGELVWCTEGDE SEQ ID NO: 75  63 nM EEPDCSWHLGELVMCTPMEV SEQ ID NO: 76  14 nM KEADCAWHMGELVWCSEMEG SEQ ID NO: 77  66 nM EQADCAWHLGELVWCTPMVF SEQ ID NO: 78   8 nM EEPDCSWHLGELVWCTPIEV SEQ ID NO: 79  15 nM GEPDCAWHLGELVWCTPMVF SEQ ID NO: 80   7 nM GEQDCSYHMGELVWCTTVDG SEQ ID NO: 81  210 nM GVRNCAYHLGELVWCTPMEF SEQ ID NO: 82  10 nM RVADCAWHMGELVWCSELEV SEQ ID NO: 83  44 nM GEADCAWHLGELVWCTPMDL SEQ ID NO: 84 N/A GEQDCSWHLGELVWCTPMEV SEQ ID NO: 85 N/A GMRDCSYHLGELVWCSDMEL SEQ ID NO: 86 N/A EVADCSWHLGELVWCTEGEF SEQ ID NO: 87  54 nM GEEDCAWHLGELVWCTDVED SEQ ID NO: 88  52 nM EVEDCAYHLGELVWCSDLEG SEQ ID NO: 89  82 nM WEEDCAWHLGELVWCAEFDE SEQ ID NO: 90  44 nM KEASCAWHLGELVWCSEVEE SEQ ID NO: 91  130 nM ALA Scan on Phage AEADCAWHLGELVWCTKVEE SEQ ID NO: 92  20 nM WAADCAWHLGELVWCTKVEE SEQ ID NO: 93  34 nM WEPDCAWHLGELVWCTKVEE SEQ ID NO: 94  36 nM WEAACAWHLGELVWCTKVEE SEQ ID NO: 95  55 nM WEAACSWHLGELVWCTKVEE SEQ ID NO: 96  10 nM WEADCAAHLGELVWCTKVEE SEQ ID NO: 97  798 nM WEADCAWALGELVWCTKVEE SEQ ID NO: 98  139 nM WEADCAWHAGELVWCTKVEE SEQ ID NO: 99  56 nM WEADCAWHLAELVWCTKVEE SEQ ID NO: 100  12 nM WEADCAWHLGALVWCTKVEE SEQ ID NO: 101  11 nM WEADCAWHLGEAVWCTKVEE SEQ ID NO: 102 1890 nM WEADCAWHLGELAWCTKVEE SEQ ID NO: 103 4670 nM WEADCAWHLGELVACTKVEE SEQ ID NO: 104 3380 nM WEADCAWHLGELVWCAKVEE SEQ ID NO: 105  101 nM WEADCAWHLGELVWCTAVEE SEQ ID NO: 106  10 nM WEADCAWHLGELVWCTKAEE SEQ ID NO: 107   8 nM WEADCAWHLGELVWCTKVAE SEQ ID NO: 108   4 nM

EXAMPLE 2

[0162] Construction of Anti-VEGF Fabs Tagged with IgG-Fc Peptide Ligands

[0163] IgG-Fc peptide ligands may be combined with a bioactive compound to form a hybrid molecule that comprises a peptide ligand domain and an active domain. In this Example, IgG-Fc peptide ligands are combined with a Fab fragment that recognizes human VEGF. A neutralizing antibody to human VEGF has been previously identified from murine hybridoma, humanized, and optimized by phage display. See Muller et al. (1998), Structure 6:1153-1167; Chen et al. (1999), J. Mol. Biol. 293:865-881; and International Patent Publication No. WO 98/45331. Two humanized Fab forms of this antibody were chosen to test whether binding affinity to an irrelevant IgG could be added to the Fabs without disrupting their antigen-binding affinity. An IgG-Fc peptide ligand, DCAWHLGELVWCT (SEQ ID NO: 8), identified and optimized by the peptide-phage display method described in Example 1 was used, along with a short peptide linker (Gly-Gly-Gly) to provide flexibility between the peptide and the Fab. The light chain of the Fab was chosen for fusions because in the case of this antibody, the light chain is known to have little contribution to antigen binding (Muller et al., 1998, supra). In principle the peptide ligand domain could function to introduce IgG-binding whether introduced at the N-terminus, C-terminus, or inserted within the original Fab sequence. Described here are N-terminal fusions DCAWHLGELVWCTGGG-(light chain) (SEQ ID NO: 109) as well as C-terminal fusions (light chain)-GGGWEADCAWHLGELVWCT (SEQ ID NO: 110).

[0164] An oligodeoxynucleotide, HL-569, was designed and synthesized for mutation of anti-VEGF plasmids to create fusions of the IgG-Fc peptide ligand at the N-terminus of the antibody light chain. The sequence of HL-569 (with added peptide sequence underlined) is: 5-ACA AAC GCG TAC GCT GAC TGC GCT TGG CAC CTG GGC GAG CTG GTC TGG TGC ACC GGA GGA GGA GAT ATC CAG TTG ACC-3′ (SEQ ID NO: 111). The GAC codon follows the STII secretion-signal sequence at the N-terminus of the light chain, and the GAT codon corresponds to the first residue of the mature (wild-type) light chain.

[0165] Another oligodeoxynucleotide, HL-570, was designed and synthesized for construction of peptide ligand fusions to the C-terminus of the antibody light chain. The sequence of HL-570 (with added peptide sequence underlined) is: 5′-AAC AGG GGA GAG TGT GGA GGA GGA TGG GAA GCA GAC TGC GCT TGG CAC CTG GGC GAG CTG GTC TGG TGC ACC TAA GCT GAT CCT CTA C-3′ (SEQ ID NO: 112). The TGT codon preceding the underscored GGA codon corresponds to residue Cys-214 of the light chain, and the TAA “stop codon” marks the end of the translated peptide sequence. Phagemids pY0192 and pY0317 (described Muller et al., 1998, supra; Chen et al., 1999; and International Patent Publication No. WO 98/45331, encoding low-affinity and high-affinity forms of a humanized anti-VEGF antibody, respectively, were mutated with each of the two IgG-peptide oligos to yield constructs pY0192-569, pY0192-570, pY0317-569, and pY0317-570.

EXAMPLE 3 Phage-ELISA Analysis of Hybrid Molecules Comprising Peptide-Ligand Tagged Anti-VEGF Fabs

[0166] A phage-ELISA competitive binding assay (Lowman (1998), Methods Mol. Biol. 87:249-264) was used to compare the apparent binding affinities of anti-VEGF antibody variants tagged with an IgG-Fc peptide ligand at their N-terminus or C-terminus and displayed monovalently on bacteriophage M13 particles as fusions to the C-terminal domain of the gene III protein.

[0167] An irrelevant humanized IgG, 4D5-IgG, also known as Herceptin®, was coated onto Nunc Maxisorp immunosorbant plates at 2 microg/mL in phosphate buffered saline solution (PBS). Phagemid particles from overnight cultures of XL1 Blue E. coli (Stratagene) were diluted in PBS containing 0.5% bovine serum albumin and 0.05% Tween-20. The phagemid particles were mixed with serial dilutions of Herceptin® in solution, equilibrated for 20 min in a non-adsorbent plate (Nunc F96), then transferred to the Herceptin®-coated Maxisorp plate for detection of unbound phage. After 20 min, the plate was washed with PBS/Tween, and developed with an anti-phage monoclonal antibody-HRP conjugate (Pharmacia) and OPD substrate (Sigma). Displacement curves (FIG. 1) showed IC₅₀ values of about 100-300 nM for each of the constructs, pY0192-569, pY0192-570, pY0317-569, and pY0317-570.

EXAMPLE 4 BIAcore™ Analysis of IgG Binding to Anti-VEGF Fab Tagged with an IgG-Fc Peptide Ligand

[0168] A surface plasmon resonance instrument (BIAcore, Inc., Piscataway, N.J.) was used to measure binding of an irrelevant IgG, 4D5-IgG, also known as Herceptin®, to Fab that previously had been bound to an immobilized VEGF biosensor chip.

[0169] Fab variants encoded by pY0317 and pY0317-570 (control anti-VEGF high affinity, humanized Fab, and anti-VEGF high affinity, humanized Fab tagged with an IgG-Fc peptide ligand domain, respectively; see Example 2, supra, and WO 98/45331) were expressed in E. coli and purified by protein-G (Pharmacia) affinity chromatography. Recombinant human VEGF was immobilized onto BIAcore™ CM-5 biosensor chips (BIAcore, Inc.) as described (Muller et al., 1998, supra). After VEGF immobilization, the chip was blocked with ethanolamine, and the peptide-ligand tagged Y0317-570 Fab, or Y0317 control, was injected in PBS buffer containing 0.05% Tween-20 and 0.01% sodium azide. Following Fab injection, Herceptin® was injected, and the dissociation off-rate (k_(off)) following injection was observed.

[0170] The results (FIG. 2) show that Herceptin® bound to the tagged but not to the control Y0317 Fab. Using a 1:1 Langmuir binding model (Karlsson et al. (1991), J. Immunuol. Methods 145:229-240 (1991)), a k_(off) of 2.8×10⁻³, sec⁻¹, and a corresponding dissociation half-life (t_(1/2)) of 8.5 min were calculated for Y0317-570. Limitations of material prevented reliable on-rate determinations. However, the observed k_(off) suggests an equilibrium binding affinity, K_(d), of 30 nM to 300 nM (assuming k_(on) of 10⁴-10⁵ M⁻¹ sec⁻¹), consistent with peptide binding and phage-ELISA results (above). Importantly, the BIAcore™ results (FIG. 2) also show that the tagged Fab can simultaneously binding both antigen (immobilized VEGF) and an irrelevant IgG.

EXAMPLE 5 IgG-Fc Peptide Ligand Tagged Anti-VEGF Fabs have Prolonged Elimination Half Times

[0171] The blood clearance rates and tissue distribution of the IgG-Fc peptide ligand-tagged anti-VEGF Fab (Fab-Y0317-570) are compared to those of the untagged control anti-VEGF Fab Y0317. Determinations of the elimination half time and volume of distribution are made in New Zealand White Rabbits of 2.8 to 3 kg weight. The amount of test article present in the plasma samples is determined using any method known in the art, such as, e.g., ELISA, or RIA.

[0172] Pharmacokinetic analysis is performed using the test article plasma concentrations. Group mean plasma data for each test article conforms to a multi-exponential profile when plotted against the time post-dosing. The data are fit by a standard two-compartment model with bolus input and first-order rate constants for distribution and elimination phases. The general equation for the best fit of the data for i.v. administration is: c(t)=Ae^(αt)+Be^(−βt), where c(t) is the plasma concentration at time t, A and B are intercepts on the Y-axis, and α and β are the apparent first-order rate constants for the distribution and elimination phases, respectively. The α-phase is the initial phase of the clearance and reflects distribution of the protein into all extracellular fluid of the animal, whereas the second or β-phase portion of the decay curve represents true plasma clearance. Methods for fitting such equations are well known in the art. For example, A=D/V(α−k21)/(α−β), B=D/V (β−k21)/(α−β), and α and β (for α>β) are roots of the quadratic equation: r²+(k12+k21+k10)r+k21k10=0 using estimated parameters of V=volume of distribution, k10=elimination rate, k12=transfer rate from compartment 1 to compartment 2 and k21=transfer rate from compartment 2 to compartment 1, and D=the administered dose.

[0173] On the morning of the study six New Zealand White rabbits (body weight 2.8-3.0 kg) were placed in restrainers. Catheters were installed in an ear artery for blood sample collection and in a contralateral ear vein for dosing.

[0174] Rabbits were divided into two groups (n=3/group). Group 1 animals received and IV bolus of control anti-VEGF Fab-Y0317. Rabbits in Group 2 received Fab-Y0317-570. A summary of group assignment and dosing information is presented in the table below. Nominal Dose Weight Dose Dose Conc. Vol. Group (kg) Dose Group (mg/kg) (mg/mL) (mL) 1 2.9 Control-Fab-Y0317 1 3 0.97 1 3.0 Control-Fab-Y0317 1 3 1.00 1 2.9 Control-Fab-Y0317 1 3 0.97 2 2.8 Fab-Y0317-570 1 3 0.93 2 3.0 Fab-Y0317-570 1 3 1.00 2 2.9 Fab-Y0317-570 1 3 0.97

[0175] Serial blood samples (0.5 mL) were collected just prior to dosing and at 10, 20 40 min, 1, 2, 3, 4, 6, 8, 24 and 48 hr after dose administration. Blood was collected in serum separator tubes, allowed to clot (˜30 min) at room temperature, and centrifuged. Serum was harvested and immediately stored at −70 C until analyzed.

[0176] ELISA plates were coated with 0.5 microg/ml VEGF in 50 mM carbonate buffer, pH 9.6, at 4° C. overnight and blocked with 0.5% bovine serum albumin, 10 ppm Proclin 300 (Supelco, Bellefonte, Pa.) in PBS (8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 2.7 mM KCl and 137 mM NaCl, pH 7.2) at room temperature for 1 hour. Standards (0.41-100 ng/ml) and twofold serial dilutions of samples (minimum dilution 1:100) in PBS containing 0.5% bovine serum albumin, 0.05% polysorbate 20, 0.25% CHAPS, 0.2% bovine gamma globulins (Sigma, St. Louis, Mo.) and 5 mM EDTA were incubated on the plates for 2 hours. Bound antibody was detected using peroxidase labeled goat F(ab′)₂ anti-human IgG F(ab′)₂ (Jackson ImmunoResearch, West Grove, Pa.), followed by 3,3′,5,5′-tetramethyl benzidine (Kirkegaard & Perry Laboratories) as the substrate. Plates were washed between steps. Absorbance was read at 450 nm on a Titerek stacker reader (ICN, Costa Mesa, Calif.). The standard curve was fitted using a four-parameter regression curve-fitting program (Kaleidagraph, Synergy Software, Reading, Pa.). Data points which fell in the range of the standard curve were used for calculating the Fab concentrations in samples.

[0177] Data analysis: Graphs of concentration versus time profiles were made using KaleidaGraph (KaleidaGraph™ V. 3.09 Copyright 1986-1997. Synergy Software. Reading, Pa.). Values reported as less than reportable. (LTR) were not included in the PK analysis and are not represented graphically. Pharmacokinetic parameters were determined by compartmental analysis using WinNonlin software (WinNonlin® Professional V. 3.1 WinNonlin™ Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif.). Pharmacokinetic parameters were computed as described elsewhere (Ritschel W A and Kearns G L. Handbook of basic pharmacokinetics including clinical applications, 5th edition. American Pharmaceutical Assoc., Washington, D.C. Copyright 1999).

[0178] The results are reported in FIG. 3. A two-compartment model with bolus input and first-order output (WinNonlin) was used to fit observed serum concentration vs. time data. Calculated pharmacokinetic parameters ware presented in the table below. Pharmacokinetic Parameter Summary (IV bolus; 1 mg/kg) Group 1 Group 2 Parameter Control Fab-Y0317 Fab-Y0317-570 AUC (h*μg/mL) 13.6 ± 1.2  215 ± 56 Cmax (μg/mL) 15.6 ± 0.6   13 ± 0.7 CL (mL/h/kg) 74.2 ± 6.7  4.8 ± 1.1 K10 half-life (hr)  0.6 ± 0.02 11.3 ± 3.6 alpha half-life (hr) 0.39 ± 0.03 1.15 ± 0.31 beta half-life (hr) 1.93 ± 0.27 37.6 ± 19 Vl (mL/kg) 64.1 ± 2.37 75.2 ± 4.23 Vss (mL/kg)  112 ± 7.7  225 ± 54

[0179] The initial volume of distribution (V1) for both agents was approximately equal to serum volume. The estimated steady state volume of distribution (Vss) for Fab-Y0317-570 (225 mL/kg) was approximately 2 fold higher than estimated for the control Fab (112 mL/kg) suggesting a significant amount of binding to endogenous IgG. Control Fab-Y0317 was eliminated approximately 15-fold faster from the serum (clearance=74 mL/h/kg) compared to Fab-Y0317-570 (4.8 mL/h/d). The overall exposure (AUC) of Fab-Y0317-570 was 16 times higher than for Fab-Y0317. Fab-Y0317 was undetectable in the serum 24 h after dosing but serum concentrations of Fab-Y0317-570 were still above 1 μg/mL 48 h after dosing. Both the distribution (alpha) half-life (1.15 h) and the elimination (beta) half-life (37.6 h) were significantly longer than the control Fab.

[0180] These results suggest that addition of a 13 amino acid that binds to endogenous IgG to Fab-Y0317 can significantly slow Fab clearance, increase half-life and enhance overall exposure.

EXAMPLE 6 Serum Albumin Peptide Ligands

[0181] Phage Libraries and Selection Conditions-Phage-displayed peptide libraries were selected against rabbit, rat and human albumin. Phage libraries expressing random peptide sequences fused to gene 8 (Lowman et al., Biochem. 37, 8870 (1998)) were pooled into 5 groups: Pool A contained CX₂GPX₄C (SEQ ID NO: 133), X₄CX₂GPX₄CX₄ (SEQ ID NO: 134) and X_(i)CX_(j)CX_(k) where j=8-10; Pool B contained X₂₀ and X_(i)CX_(j)CX_(k) where j=4-7; Pool C contained X₈ and X₂CX_(j)CX₂ where j=4-6; Pool D contained X₂CX_(j)CX₂ where j=7-10; Pool E contained CX₆CX₆CCX₃CX₆C (SEQ ID NO: 135), CCX₃CX₆C (SEQ ID NO: 136), CCX₅CX₄CX₄CC (SEQ ID NO: 137), CXCX₇CX₃CX₆ (SEQ ID NO: 138) where X represents any of the 20 naturally occurring L-amino acids. In each case i+j+k=18 and |i−k|<2. Each of the 10 libraries has in excess of 108 clones.

[0182] The phage library pools were suspended in binding buffer (PBS, 1% ovalbumin, 0.005% Tween 20) and sorted against rabbit, rat or human albumin immobilized directly on maxisorp plates (10 μg/ml in PBS, overnight at 4° C.; plates were blocked with Blocker Casein (Pierce Chemical, Rockford, Ill.)). After 2 hours, unbound phage were removed by repetitive washing (PBS, 0.05% Tween 20) and bound phage were eluted with 500 mM KCl, 10 mM HCl, pH 2. Eluted phage were propagated in XL1-Blue cells with VCSM13 helper phage (Stratagene, La Jolla, Calif.). Enrichment was monitored by titering the number of phage that bound to an albumin coated well compared to a well coated with ovalbumin or casein.

[0183] Phage ELISA—Phage clones (˜10¹¹ phage) were added to plates coated with rat, rabbit or human albumin. The microtiter plate was washed with wash buffer and bound phage were detected with HRP/Anti-M13 Conjugate. The amount of HRP bound was measured using ABTS/H₂O₂ substrate and monitoring the change at 405 nm.

[0184] The peptide sequences displayed by phage clones selected for binding to rabbit, human or rat albumin are shown in FIG. 4. Also indicated is the ability of individual phage clones to bind the 3 species of immobilized albumin. This was tested using a phage ELISA. Note that clone RB, selected for binding to rat albumin is also capable of binding human and rabbit albumin.

[0185] Sequence Maturation on Monovalent Phage—Partially randomized libraries were designed using oligonucleotides coding for each of the selected clones in FIG. 4, but synthesized with a 70-10-10-10 mixture of bases as described (Dennis et al., Nature 404, 465 (2000)). Although the potential diversity of these libraries is the same as the initial naïve libraries, each ‘soft randomized’ library maintains a bias towards the selected sequence in FIG. 4. Each library was again selected for binding to rat, rabbit or human albumin regardless of its origin. For example, the library resulting from soft randomization of clone RB was selected against rat, rabbit or human albumin even though it was originally identified for binding to rat albumin. Sequences identified following soft randomization are shown in FIG. 5 along with their species specificity as determined by phage ELISA. Most clones appear to be specific for the species of albumin for which they were selected, however, clones from the RB soft randomization library bind to all three species.

[0186] Phage clones were also tested for binding to rhesus, mouse and bovine albumin. Clones originating from the RB soft randomization library were found to bind each of these species of albumin as well and were specific for albumin based upon their lack of binding to ovalbumin and casein (FIG. 6). Clones that bind to multiple species of albumin (multi-species binders) are listed in FIG. 7.

[0187] Hard randomization—Sequences from soft randomization of the RB sequence were further matured using a hard randomization strategy. A new library was designed that kept highly selected residues (underlined) constant X₅DXCLPXWGCLWX₄ (SEQ ID NO: 116), while fully randomizing the remaining positions. A second library, one residue shorter at both the N and C terminus was also constructed. Sequences from these libraries selected against rat, rabbit and human albumin are shown in FIGS. 8A, 8B, and 8C respectively.

[0188] Peptide Synthesis-Peptides were synthesized by either manual or automated (Milligen 9050) Fmoc-based solid (phase synthesis on a 0.25 mmol scale using a PEG-polystyrene resin (Bodanszky M., (1984) Principles of Peptide Synthesis, Springer, Berlin). Side chain protecting groups were removed and the peptides were cleaved from the resin with 95% trifluoroacetic acid (TFA) and 5% triisopropylsilane. A saturated iodine solution in acetic acid was added for oxidation of disulfide bonds. Peptides were purified by reversed phase HPLC using a water/acetonitrile gradient containing 0.1% TFA. Peptides were >95% pure by analytical HPLC and its identity verified by mass spectrometry.

[0189] The carboxy terminal lysine of peptide SA08 was derivatized with NHS-LC-biotin (Pierce Chemical, Rockford, Ill.) and purified by HPLC as above yielding SA08b (Ac-QGLIGDICLPRWGCLWGDSVK_(b) (SEQ ID NO: 124)-n where K_(b) refers to lysine-biotin).

[0190] SA08b Binding Assay—Rabbit, rat or mouse albumin was immobilized directly on maxisorp plates at 10 μg/ml in PBS, overnight at 4° C. Plates were blocked using Blocker Casein (Pierce Chemical, Rockford, Ill.) for 1 hr, at 25° C. Serially diluted samples were suspended in binding buffer (above) and added to the plate followed by the addition of 10 nM SA08b for 1 hr, at 25° C. The microtiter plate was washed with PBS, 0.05% Tween 20 and the SA08b bound to albumin was detected with Streptavidin/HRP. The amount of HRP bound was measured using ABTS/H₂O₂ substrate and monitoring the change at 405 nm.

[0191] Peptides corresponding to identified phage sequences were synthesized and their affinity for rat, rabbit or mouse albumin measured using the SA08b binding assay (FIGS. 9 and 10).

[0192] Construction, Expression and Purification of Albumin Binding Fab Fusions—In order to test whether association with albumin could increase the half-life of proteins and peptides in vivo, the sequence of SA06 was fused to a Fab fragment (D3H44) directed for binding tissue factor (TF). The SA06 sequence was added to the carboxy terminus of either the light chain (D3H44-L) or heavy chain (D3H44-Ls) of the Fab. In addition, as a precaution against folding problems, identical constructions were made but with the intra-chain disulfide replaced by alanines (D3H44-Ls and D3H44Hs, respectively) as depicted in FIG. 11.

[0193] The fusions were expressed under control of the alkaline phosphatase promoter and secreted from E. coli using the stII secretion signal. Fab fusions were recovered from the periplasm by suspending cells in 1 mM EDTA, 10 mM Tris-HCl, pH8, for 1 hr at 4° C. Cell debris was removed by centrifugation and the anti-TF Fab was selectively purified using a Hi-Trap (Amersham Pharmacia Biotech, Piscataway, N.J.) TF affinity column. Properly folded D3H44L or D3H44-Ls was further purified using a rabbit albumin affinity column (rabbit albumin coupled to CNBr-activated Sepharose 4B, Amersham Pharmacia Biotech, Piscataway, N.J.). Both columns were washed with PBS and eluted with 50 mM HCl. Eluted fractions were neutralized with 1 M Tris pH 8. Endotoxin was further removed following extraction with triton X114 (Aida and Pabst, J. Immunol. Methods 132, 191 (1990)).

[0194] Purified D3H44 fusions retained their ability to bind TF as measured using a FX activation assay (FIG. 12), and a prothrombin time assay that measures prolongation of tissue factor dependent clotting (FIG. 13)(for methods see Dennis et al., Nature 404, 465 (2000)). Unlike D3H44 lacking the albumin binding sequence (WT), both D3H44-L and D3H44-Ls are able to bind to albumin as measured in the SA08b binding assay (FIG. 14). Further, both D3H44 albumin-binding fusions are capable of binding TF and albumin simultaneously as judged by a biotin-TF binding assay (FIG. 15). In this assay, the binding of the D3H44 fusions to immobilized albumin is detected with biotinylated TF. Wild-type D3H44 (WT) is unable to bind albumin and thus does not generate a signal upon addition of biotinylated TF.

[0195] Pharmacokinetics of D3H44 albumin-binding fusions—D3H44 variants were given as a 0.5 mg/kg bolus in rabbit. Each group consisted of 3 rabbits (5 in the F(ab′)₂ group). Serum samples taken at the indicated time points were serially diluted and the concentration of D3H44 determined using a TF binding ELISA.

[0196] Pharmacokinetic analysis is performed using the test article plasma concentrations. Group mean plasma data for each test article conforms to a multi-exponential profile when plotted against the time post-dosing. The data are fit by a standard two-compartment model with bolus input and first-order rate constants for distribution and elimination phases. The general equation for the best fit of the data for i.v. administration is: c(t)=Ae^(−αt)+Be^(−βt), where c(t) is the plasma concentration at time t, A and B are intercepts on the Y-axis, and α and β are the apparent first-order rate constants for the distribution and elimination phases, respectively. The α-phase is the initial phase of the clearance and reflects distribution of the protein into all extracellular fluid of the animal, whereas the second or β-phase portion of the decay curve represents true plasma clearance. Methods for fitting such equations are well known in the art. For example, A=D/V(α−k21)/(α−β), B=D/V (β−k21)/(α−β), and α and β (for α>β) are roots of the quadratic equation: r²+(k12+k21+k10)r+k21k10=0 using estimated parameters of V=volume of distribution, k10=elimination rate, k12=transfer rate from compartment 1 to compartment 2 and k21=transfer rate from compartment 2 to compartment 1, and D=the administered dose.

[0197] Data analysis: Graphs of concentration versus time, profiles were made using KaleidaGraph (KaleidaGraph™ V. 3.09 Copyright 1986-1997. Synergy Software. Reading, Pa.). Values reported as less than reportable (LTR) were not included in the PK analysis and are not represented graphically. Pharmacokinetic parameters were determined by compartmental analysis using WinNonlin software (WinNonlin® Professional V. 3.1 WinNonlin™ Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif.). Pharmacokinetic parameters were computed as described elsewhere (Ritschel W A and Kearns GL. Handbook of basic pharmacokinetics including clinical applications, 5th edition. American Pharmaceutical Assoc., Washington, D.C. Copyright 1999).

[0198] Fusion of the albumin binding peptide to D3H44 results in a protein having improved pharmacokinetic parameters (FIGS. 16 and 17). D3H44L has a 70-fold increase in half-life (K10-HL) relative to wild-type Fab and a comparable half-life to D3H44 Fabs derivatized with 20K or 40K polyethylene glycol (PEG).

[0199] All publications cited herein are expressly incorporated by reference in their entirety.

1 492 1 7 PRT Artificial Sequence Synthesized 1 Gly Gly Gly Ser Gly Gly Gly 1 5 2 20 PRT Artificial Sequence Synthesized 2 Glu Thr Gln Arg Cys Thr Trp His Met Gly Glu Leu Val Trp Cys Glu 1 5 10 15 Arg Glu His Asn 20 3 20 PRT Artificial Sequence Synthesized 3 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Val 1 5 10 15 Ala Gly Val Glu 20 4 7 PRT Artificial Sequence Synthesized 4 Gly Gly Gly Pro Gly Gly Gly 1 5 5 60 DNA Artificial Sequence Synthesized 5 drggwagmar rctgckcttr scacmtgggc gagctggtct ggtgcrvcrv mbkcgaskdw 60 6 60 DNA Artificial Sequence Synthesized 6 drsvwgsvgr rctgckcctr syrsmtgggc gagctggtct ggtgcrncvv snbsgwskdm 60 7 60 DNA Artificial Sequence Synthesized 7 dnsnnsnnsv nstgcbvgtd shrsmdsggc gagstckkgw rgtgcrnmnn snnsnnsnnm 60 8 13 PRT Artificial Sequence Synthesized 8 Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 9 9 PRT Artificial Sequence Synthesized 9 Xaa Xaa Xaa Xaa Xaa Xaa Leu Val Trp 1 5 10 9 PRT Artificial Sequence Synthesized 10 Xaa Xaa Xaa Xaa Gly Glu Leu Val Trp 1 5 11 20 PRT Artificial Sequence Synthesized 11 Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Leu Val Trp Cys Xaa 1 5 10 15 Xaa Xaa Xaa Xaa 20 12 20 PRT Artificial Sequence Synthesized 12 Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Gly Glu Leu Val Trp Cys Xaa 1 5 10 15 Xaa Xaa Xaa Xaa 20 13 20 PRT Artificial Sequence Synthesized 13 Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Gly Glu Leu Val Trp Cys Xaa 1 5 10 15 Xaa Xaa Xaa Xaa 20 14 20 PRT Artificial Sequence Synthesized 14 Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Gly Glu Leu Val Trp Cys Xaa 1 5 10 15 Xaa Xaa Xaa Xaa 20 15 20 PRT Artificial Sequence Synthesized 15 Asp Leu Ala Asp Cys Ser Trp His Met Gly Glu Leu Val Trp Cys Ser 1 5 10 15 Arg Val Glu Gly 20 16 20 PRT Artificial Sequence Synthesized 16 Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Pro Met Glu Phe 20 17 20 PRT Artificial Sequence Synthesized 17 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Asp 1 5 10 15 Thr Leu Thr Glu 20 18 20 PRT Artificial Sequence Synthesized 18 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Ser 1 5 10 15 Pro Gly Val Glu 20 19 20 PRT Artificial Sequence Synthesized 19 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Ser 1 5 10 15 Gly Val Glu Gly 20 20 20 PRT Artificial Sequence Synthesized 20 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Ser 1 5 10 15 Ala Gly Val Glu 20 21 20 PRT Artificial Sequence Synthesized 21 Glu Ser Glu Asp Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Val 1 5 10 15 Ala Gly Val Glu 20 22 20 PRT Artificial Sequence Synthesized 22 Glu Lys Glu Asp Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Val 1 5 10 15 Ala Gly Val Glu 20 23 20 PRT Artificial Sequence Synthesized 23 Glu Asp Pro Asp Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Val 1 5 10 15 Ala Gly Val Glu 20 24 20 PRT Artificial Sequence Synthesized 24 Glu Glu Ala Asp Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Val 1 5 10 15 Ala Gly Val Glu 20 25 20 PRT Artificial Sequence Synthesized 25 Asn Ala Asp Asp Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Val 1 5 10 15 Ala Gly Val Glu 20 26 20 PRT Artificial Sequence Synthesized 26 Ser Glu Thr Thr Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Val 1 5 10 15 Ala Gly Val Glu 20 27 19 PRT Artificial Sequence Synthesized 27 Ala Trp Lys Thr Cys Gln Trp Leu Gly Glu Leu Val Trp Cys Val Ala 1 5 10 15 Gly Val Glu 28 20 PRT Artificial Sequence Synthesized 28 Asp Leu Ala Asp Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Ser 1 5 10 15 Arg Val Glu Gly 20 29 20 PRT Artificial Sequence Synthesized 29 Lys Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Val 1 5 10 15 Ala Gly Val Glu 20 30 20 PRT Artificial Sequence Synthesized 30 Lys Glu Ala Glu Cys Ser Tyr His Leu Gly Glu Leu Val Trp Cys Val 1 5 10 15 Ala Gly Val Glu 20 31 20 PRT Artificial Sequence Synthesized 31 Lys Glu Ala Arg Cys Trp Tyr Trp His Gly Glu Leu Val Trp Cys Ser 1 5 10 15 Asp Pro Glu Glu 20 32 20 PRT Artificial Sequence Synthesized 32 Lys Glu Ala Ser Cys Ser Tyr His Leu Gly Glu Leu Val Trp Cys Val 1 5 10 15 Ala Gly Val Glu 20 33 20 PRT Artificial Sequence Synthesized 33 Lys Glu Ala Ser Cys Ser Trp His Leu Gly Glu Leu Val Trp Cys Val 1 5 10 15 Ala Gly Val Glu 20 34 20 PRT Artificial Sequence Synthesized 34 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Glu Gly Val Glu 20 35 20 PRT Artificial Sequence Synthesized 35 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Asp 1 5 10 15 Asp Gly Val Glu 20 36 20 PRT Artificial Sequence Synthesized 36 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Ser 1 5 10 15 Glu Gly Val Glu 20 37 20 PRT Artificial Sequence Synthesized 37 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Val Trp Lys Cys Lys 1 5 10 15 Ser Gly Val Glu 20 38 20 PRT Artificial Sequence Synthesized 38 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Asp 1 5 10 15 Asn Gly Val Glu 20 39 20 PRT Artificial Sequence Synthesized 39 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Asp 1 5 10 15 Thr Phe Asp Glu 20 40 20 PRT Artificial Sequence Synthesized 40 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Asp 1 5 10 15 Gly Leu Asp Glu 20 41 20 PRT Artificial Sequence Synthesized 41 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Val 1 5 10 15 Gly Leu Asp Glu 20 42 20 PRT Artificial Sequence Synthesized 42 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Glu 1 5 10 15 Asp Thr Leu Glu 20 43 20 PRT Artificial Sequence Synthesized 43 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Glu 1 5 10 15 Asp Thr Met Glu 20 44 20 PRT Artificial Sequence Synthesized 44 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Glu 1 5 10 15 Asp Met Met Glu 20 45 20 PRT Artificial Sequence Synthesized 45 Trp Val Glu Asp Cys Ser Trp His Met Gly Glu Leu Val Trp Cys Asp 1 5 10 15 Gly Gly Glu Phe 20 46 20 PRT Artificial Sequence Synthesized 46 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Asp 1 5 10 15 Trp Met Asn Gly 20 47 20 PRT Artificial Sequence Synthesized 47 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Asp 1 5 10 15 Asp Thr Pro Val 20 48 20 PRT Artificial Sequence Synthesized 48 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Asp 1 5 10 15 Asp Tyr Gly Glu 20 49 20 PRT Artificial Sequence Synthesized 49 Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Ser 1 5 10 15 Asp Leu Trp Glu 20 50 20 PRT Artificial Sequence Synthesized 50 Trp Arg Gly Gly Cys Ser Trp His Met Gly Glu Leu Val Trp Cys Glu 1 5 10 15 His Asp Met Glu 20 51 20 PRT Artificial Sequence Synthesized 51 Ala Val Ser Lys Cys Ser Phe His Met Gly Glu Leu Val Trp Cys Ser 1 5 10 15 Asp Val Met Asn 20 52 20 PRT Artificial Sequence Synthesized 52 Asn Gln Val Ser Cys Ser Tyr Ser Arg Gly Glu Leu Val Trp Cys Ser 1 5 10 15 Lys Gln Ser Gln 20 53 20 PRT Artificial Sequence Synthesized 53 Gly Arg Met Glu Cys Ala Trp His Gln Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Pro Thr Leu Glu 20 54 20 PRT Artificial Sequence Synthesized 54 Gly Thr Met Glu Cys Ser Trp His Gln Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Pro Thr Leu Ala 20 55 20 PRT Artificial Sequence Synthesized 55 Glu Met Arg Asp Cys Ser Trp His Leu Gly Glu Leu Val Trp Cys Ala 1 5 10 15 His Met Glu Gly 20 56 20 PRT Artificial Sequence Synthesized 56 Gly Ser Trp Glu Cys Ala Tyr His Leu Gly Glu Leu Val Trp Cys Glu 1 5 10 15 Thr Gly Ser Gly 20 57 20 PRT Artificial Sequence Synthesized 57 Val Ala Glu Pro Cys Ala Tyr His Leu Gly Glu Leu Val Trp Cys Glu 1 5 10 15 Val Leu Lys Gly 20 58 20 PRT Artificial Sequence Synthesized 58 Lys Glu Ala Met Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Glu 1 5 10 15 Ser Asp Met Pro 20 59 20 PRT Artificial Sequence Synthesized 59 Asp Leu Ala Asp Cys Ser Trp His Leu Gly Glu Leu Val Trp Cys Ser 1 5 10 15 Arg Val Glu Gly 20 60 20 PRT Artificial Sequence Synthesized 60 Asp Leu Ala Asp Cys Ser Trp His Leu Gly Glu Leu Val Trp Cys Val 1 5 10 15 Gly Leu Asp Glu 20 61 20 PRT Artificial Sequence Synthesized 61 Trp Val Glu Asp Cys Ser Trp His Leu Gly Glu Leu Val Trp Cys Val 1 5 10 15 Gly Leu Asp Phe 20 62 20 PRT Artificial Sequence Synthesized 62 Lys Val Ala Asp Cys Ala Trp His Met Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Glu Val Glu Gly 20 63 20 PRT Artificial Sequence Synthesized 63 Gly Glu Glu Asp Cys Ser Tyr His Leu Gly Glu Leu Val Met Cys Thr 1 5 10 15 Glu Leu Asp Asp 20 64 20 PRT Artificial Sequence Synthesized 64 Gly Val Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Glu Arg Glu Asp 20 65 20 PRT Artificial Sequence Synthesized 65 Gly Glu Glu Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Ser 1 5 10 15 Gly Gly Asp Phe 20 66 20 PRT Artificial Sequence Synthesized 66 Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Lys Val Glu Glu 20 67 20 PRT Artificial Sequence Synthesized 67 Gly Glu Ala Asp Cys Ser Tyr His Leu Gly Glu Leu Val Trp Cys Asn 1 5 10 15 Asp Phe Glu Glu 20 68 19 PRT Artificial Sequence Synthesized 68 Trp Val Asp Cys Ala Tyr His Leu Gly Glu Leu Val Trp Cys Ser Thr 1 5 10 15 Phe Glu Glu 69 20 PRT Artificial Sequence Synthesized 69 Trp Val Glu Asp Cys Ala Trp His Met Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Lys Val Asp Glu 20 70 20 PRT Artificial Sequence Synthesized 70 Arg Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Ser 1 5 10 15 Glu Arg Asp Leu 20 71 20 PRT Artificial Sequence Synthesized 71 Glu Glu Ala Ser Cys Ala Tyr His Leu Gly Glu Leu Val Trp Cys Asp 1 5 10 15 Ala Phe Asp Val 20 72 20 PRT Artificial Sequence Sythesized 72 Arg Val Ala Ser Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Asp 1 5 10 15 Gly Leu Asp Gly 20 73 20 PRT Artificial Sequence Synthesized 73 Gly Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Lys Val Glu Glu 20 74 20 PRT Artificial Sequence Synthesized 74 Gly Glu Ala Ser Cys Ala Tyr His Leu Gly Glu Leu Val Trp Cys Asp 1 5 10 15 Glu Gly Glu Gly 20 75 20 PRT Artificial Sequence Synthesized 75 Arg Val Glu Asp Cys Ala Tyr His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Glu Gly Asp Glu 20 76 20 PRT Artificial Sequence Synthesized 76 Glu Glu Pro Asp Cys Ser Trp His Leu Gly Glu Leu Val Met Cys Thr 1 5 10 15 Pro Met Glu Val 20 77 20 PRT Artificial Sequence Synthesized 77 Lys Glu Ala Asp Cys Ala Trp His Met Gly Glu Leu Val Trp Cys Ser 1 5 10 15 Glu Met Glu Gly 20 78 20 PRT Artificial Sequence Synthesized 78 Glu Gln Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Pro Met Val Phe 20 79 20 PRT Artificial Sequence Synthesized 79 Glu Glu Pro Asp Cys Ser Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Pro Ile Glu Val 20 80 20 PRT Artificial Sequence Synthesized 80 Gly Glu Pro Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Pro Met Val Phe 20 81 20 PRT Artificial Sequence Synthesized 81 Gly Glu Gln Asp Cys Ser Tyr His Met Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Thr Val Asp Gly 20 82 20 PRT Artificial Sequence Synthesized 82 Gly Val Arg Asn Cys Ala Tyr His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Pro Met Glu Phe 20 83 20 PRT Artificial Sequence Synthesized 83 Arg Val Ala Asp Cys Ala Trp His Met Gly Glu Leu Val Trp Cys Ser 1 5 10 15 Glu Leu Glu Val 20 84 20 PRT Artificial Sequence Synthesized 84 Gly Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Pro Met Asp Leu 20 85 20 PRT Artificial Sequence Synthesized 85 Gly Glu Gln Asp Cys Ser Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Pro Met Glu Val 20 86 20 PRT Artificial Sequence Synthesized 86 Gly Met Arg Asp Cys Ser Tyr His Leu Gly Glu Leu Val Trp Cys Ser 1 5 10 15 Asp Met Glu Leu 20 87 20 PRT Artificial Sequence Synthesized 87 Glu Val Ala Asp Cys Ser Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Glu Gly Glu Phe 20 88 20 PRT Artificial Sequence Synthesized 88 Gly Glu Glu Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Asp Val Glu Asp 20 89 20 PRT Artificial Sequence Synthesized 89 Glu Val Glu Asp Cys Ala Tyr His Leu Gly Glu Leu Val Trp Cys Ser 1 5 10 15 Asp Leu Glu Gly 20 90 20 PRT Artificial Sequence Synthesized 90 Trp Glu Glu Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Ala 1 5 10 15 Glu Phe Asp Glu 20 91 20 PRT Artificial Sequence Synthesized 91 Lys Glu Ala Ser Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Ser 1 5 10 15 Glu Val Glu Glu 20 92 20 PRT Artificial Sequence Synthesized 92 Ala Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Lys Val Glu Glu 20 93 20 PRT Artificial Sequence Synthesized 93 Trp Ala Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Lys Val Glu Glu 20 94 20 PRT Artificial Sequence Synthesized 94 Trp Glu Pro Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Lys Val Glu Glu 20 95 20 PRT Artificial Sequence Synthesized 95 Trp Glu Ala Ala Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Lys Val Glu Glu 20 96 20 PRT Artificial Sequence Synthesized 96 Trp Glu Ala Ala Cys Ser Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Lys Val Glu Glu 20 97 20 PRT Artificial Sequence Synthesized 97 Trp Glu Ala Asp Cys Ala Ala His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Lys Val Glu Glu 20 98 20 PRT Artificial Sequence Synthesized 98 Trp Glu Ala Asp Cys Ala Trp Ala Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Lys Val Glu Glu 20 99 20 PRT Artificial Sequence Synthesized 99 Trp Glu Ala Asp Cys Ala Trp His Ala Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Lys Val Glu Glu 20 100 20 PRT Artificial Sequence Synthesized 100 Trp Glu Ala Asp Cys Ala Trp His Leu Ala Glu Leu Val Trp Cys Thr 1 5 10 15 Lys Val Glu Glu 20 101 20 PRT Artificial Sequence Synthesized 101 Trp Glu Ala Asp Cys Ala Trp His Leu Gly Ala Leu Val Trp Cys Thr 1 5 10 15 Lys Val Glu Glu 20 102 20 PRT Artificial Sequence Synthesized 102 Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Ala Val Trp Cys Thr 1 5 10 15 Lys Val Glu Glu 20 103 20 PRT Artificial Sequence Synthesized 103 Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Ala Trp Cys Thr 1 5 10 15 Lys Val Glu Glu 20 104 20 PRT Artificial Sequence Synthesized 104 Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Ala Cys Thr 1 5 10 15 Lys Val Glu Glu 20 105 20 PRT Artificial Sequence Synthesized 105 Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Ala 1 5 10 15 Lys Val Glu Glu 20 106 20 PRT Artificial Sequence Synthesized 106 Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Ala Val Glu Glu 20 107 20 PRT Artificial Sequence Synthesized 107 Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Lys Ala Glu Glu 20 108 20 PRT Artificial Sequence Synthesized 108 Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr 1 5 10 15 Lys Val Ala Glu 20 109 16 PRT Artificial Sequence Synthesized 109 Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr Gly Gly Gly 1 5 10 15 110 19 PRT Artificial Sequence Synthesized 110 Gly Gly Gly Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val 1 5 10 15 Trp Cys Thr 111 78 DNA Artificial Sequence Synthesized 111 acaaacgcgt acgctgactg cgcttggcac ctgggcgagc tggtctggtg caccggagga 60 ggagatatcc agttgacc 78 112 88 DNA Artificial Sequence Synthesized 112 aacaggggag agtgtggagg aggatgggaa gcagactgcg cttggcacct gggcgagctg 60 gtctggtgca cctaagctga tcctctac 88 113 11 PRT Artificial Sequence Synthesized 113 Phe Cys Xaa Asp Trp Pro Xaa Xaa Xaa Ser Cys 1 5 10 114 9 PRT Artificial Sequence Synthesized 114 Val Cys Tyr Xaa Xaa Xaa Ile Cys Phe 1 5 115 7 PRT Artificial Sequence Synthesized 115 Cys Tyr Xaa Pro Gly Xaa Cys 1 5 116 11 PRT Artificial Sequence Synthesized 116 Asp Xaa Cys Leu Pro Xaa Trp Gly Cys Leu Trp 1 5 10 117 12 PRT Artificial Sequence Synthesized 117 Trp Cys Asp Xaa Xaa Leu Xaa Ala Xaa Asp Leu Cys 1 5 10 118 10 PRT Artificial Sequence Synthesized 118 Asp Leu Val Xaa Leu Gly Leu Glu Cys Trp 1 5 10 119 11 PRT Artificial Sequence Synthesized 119 Asp Leu Cys Leu Arg Asp Trp Gly Cys Leu Trp 1 5 10 120 11 PRT Artificial Sequence Synthesized 120 Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 121 15 PRT Artificial Sequence Synthesized 121 Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp 1 5 10 15 122 20 PRT Artificial Sequence Synthesized 122 Gln Arg Leu Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Asp Asp Phe 20 123 20 PRT Artificial Sequence Synthesized 123 Gln Gly Leu Ile Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Ser Val 20 124 21 PRT Artificial Sequence Synthesized 124 Gln Gly Leu Ile Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Ser Val Lys 20 125 15 PRT Artificial Sequence Synthesized 125 Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp Asp 1 5 10 15 126 18 PRT Artificial Sequence Synthesized 126 Arg Leu Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Asp Asp 127 16 PRT Artificial Sequence Synthesized 127 Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp Asp 1 5 10 15 128 18 PRT Artificial Sequence Synthesized 128 Arg Leu Met Glu Asp Ile Cys Leu Ala Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Asp Asp 129 20 PRT Artificial Sequence Synthesized 129 Glu Val Arg Ser Phe Cys Thr Asp Trp Pro Ala Glu Lys Ser Cys Lys 1 5 10 15 Pro Leu Arg Gly 20 130 20 PRT Artificial Sequence Synthesized 130 Arg Ala Pro Glu Ser Phe Val Cys Tyr Trp Glu Thr Ile Cys Phe Glu 1 5 10 15 Arg Ser Glu Gln 20 131 11 PRT Artificial Sequence Synthesized 131 Glu Met Cys Tyr Phe Pro Gly Ile Cys Trp Met 1 5 10 132 5 PRT Artificial Sequence Synthesized 132 Gly Glu Leu Val Trp 1 5 133 10 PRT Artificial Sequence Synthesized 133 Cys Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Cys 1 5 10 134 18 PRT Artificial Sequence Synthesized 134 Xaa Xaa Xaa Xaa Cys Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Cys Xaa Xaa 1 5 10 15 Xaa Xaa 135 27 PRT Artificial Sequence Synthesized 135 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys 1 5 10 15 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys 20 25 136 13 PRT Artificial Sequence Synthesized 136 Cys Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 137 19 PRT Artificial Sequence Synthesized 137 Cys Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 15 Xaa Cys Cys 138 21 PRT Artificial Sequence Synthesized 138 Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa 20 139 9 PRT Artificial Sequence Synthesized 139 Xaa Xaa Xaa Xaa Gly Glu Leu Val Trp 1 5 140 11 PRT Artificial Sequence Synthesized 140 Glu Met Cys Tyr Phe Pro Gly Ile Cys Trp Met 1 5 10 141 27 PRT Artificial Sequence Synthesized 141 Cys Glu Val Ala Leu Asp Ala Cys Arg Gly Gly Glu Ser Gly Cys Cys 1 5 10 15 Arg His Ile Cys Glu Leu Ile Arg Gln Leu Cys 20 25 142 21 PRT Artificial Sequence Synthesized 142 Cys Gly Cys Val Asp Val Ser Asp Trp Asp Cys Trp Ser Glu Cys Leu 1 5 10 15 Trp Ser His Gly Ala 20 143 10 PRT Artificial Sequence Synthesized 143 Asp Leu Cys Asp Val Asp Phe Cys Trp Phe 1 5 10 144 15 PRT Artificial Sequence Synthesized 144 Asp Thr Cys Val Asp Leu Val Arg Leu Gly Leu Glu Cys Trp Gly 1 5 10 15 145 16 PRT Artificial Sequence Synthesized 145 Lys Ser Cys Ser Glu Leu His Trp Leu Leu Val Glu Glu Cys Leu Phe 1 5 10 15 146 20 PRT Artificial Sequence Synthesized 146 Met Asp Glu Leu Ala Phe Tyr Cys Gly Ile Trp Glu Cys Leu Met His 1 5 10 15 Gln Glu Gln Lys 20 147 20 PRT Artificial Sequence Synthesized 147 Arg Asn Glu Asp Pro Cys Val Val Leu Leu Glu Met Gly Leu Glu Cys 1 5 10 15 Trp Glu Gly Val 20 148 20 PRT Artificial Sequence Synthesized 148 Glu Val Arg Ser Phe Cys Thr Asp Trp Pro Ala Glu Lys Ser Cys Lys 1 5 10 15 Pro Leu Arg Gly 20 149 20 PRT Artificial Sequence Synthesized 149 Gly Glu Asn Trp Cys Asp Ser Thr Leu Met Ala Tyr Asp Leu Cys Gly 1 5 10 15 Gln Val Asn Met 20 150 20 PRT Artificial Sequence Synthesized 150 Gln Arg Gln Met Val Asp Phe Cys Leu Pro Gln Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Gly Phe 20 151 11 PRT Artificial Sequence Synthesized 151 Ala Leu Cys Tyr Phe Pro Gly Ile Cys Trp Met 1 5 10 152 15 PRT Artificial Sequence Synthesized 152 Ala Ser Glu Ile Cys Tyr Phe Pro Gly Ile Cys Trp Met Val Glu 1 5 10 15 153 11 PRT Artificial Sequence Synthesized 153 Asp Ile Cys Tyr Ile Pro Gly Ile Cys Trp Met 1 5 10 154 11 PRT Artificial Sequence Synthesized 154 Asp Leu Cys Tyr Phe Pro Gly Ile Cys Trp Met 1 5 10 155 11 PRT Artificial Sequence Synthesized 155 Asp Met Cys Tyr Phe Pro Gly Ile Cys Phe Asn 1 5 10 156 11 PRT Artificial Sequence Synthesized 156 Asp Met Cys Tyr Phe Pro Gly Ile Cys Trp Leu 1 5 10 157 15 PRT Artificial Sequence Synthesized 157 Asp Ser Glu Val Cys Tyr Phe Pro Gly Ile Cys Trp Ser Gly Thr 1 5 10 15 158 11 PRT Artificial Sequence Synthesized 158 Asp Val Cys Tyr Phe Pro Gly Ile Cys Trp Met 1 5 10 159 15 PRT Artificial Sequence Synthesized 159 Glu His Asp Met Cys Tyr Phe Pro Gly Ile Cys Trp Ile Ala Asp 1 5 10 15 160 11 PRT Artificial Sequence Synthesized 160 Glu Ile Cys Tyr Phe Pro Gly Ile Cys Trp Ile 1 5 10 161 11 PRT Artificial Sequence Synthesized 161 Glu Ile Cys Tyr Phe Pro Gly Ile Cys Trp Met 1 5 10 162 11 PRT Artificial Sequence Synthesized 162 Glu Leu Cys Tyr Phe Pro Gly Ile Cys Trp Met 1 5 10 163 11 PRT Artificial Sequence Synthesized 163 Glu Leu Cys Tyr Phe Pro Gly Ile Cys Trp Pro 1 5 10 164 11 PRT Artificial Sequence Synthesized 164 Glu Leu Cys Tyr Phe Pro Gly Ile Cys Trp Thr 1 5 10 165 11 PRT Artificial Sequence Synthesized 165 Glu Met Cys Tyr Phe Pro Gly Ile Cys Trp Ser 1 5 10 166 11 PRT Artificial Sequence Synthesized 166 Glu Met Cys Tyr Phe Pro Gly Ile Cys Trp Thr 1 5 10 167 11 PRT Artificial Sequence Synthesized 167 Glu Thr Cys Tyr Phe Pro Gly Ile Cys Trp Leu 1 5 10 168 11 PRT Artificial Sequence Synthesized 168 Glu Val Cys Tyr Phe Pro Gly Ile Cys Trp Glu 1 5 10 169 11 PRT Artificial Sequence Synthesized 169 Glu Val Cys Tyr Phe Pro Gly Ile Cys Trp Phe 1 5 10 170 11 PRT Artificial Sequence Synthesized 170 Glu Val Cys Tyr Phe Pro Gly Ile Cys Trp Met 1 5 10 171 20 PRT Artificial Sequence Synthesized 171 Glu Val Arg Ser Phe Cys Thr Asp Trp Pro Ala His Tyr Ser Cys Thr 1 5 10 15 Ser Leu Gln Gly 20 172 20 PRT Artificial Sequence Synthesized 172 Gly Glu Asp Trp Cys Asp Ser Thr Leu Leu Ala Phe Asp Leu Cys Gly 1 5 10 15 Glu Gly Ala Arg 20 173 20 PRT Artificial Sequence Synthesized 173 Gly Glu Asn Trp Cys Asp Trp Val Leu Leu Ala Tyr Asp Leu Cys Gly 1 5 10 15 Glu Asp Asn Thr 20 174 15 PRT Artificial Sequence Synthesized 174 Gly Gly Glu Ile Cys Tyr Phe Pro Gly Ile Cys Arg Val Leu Pro 1 5 10 15 175 11 PRT Artificial Sequence Synthesized 175 Gly Met Cys Tyr Phe Pro Gly Ile Cys Trp Ala 1 5 10 176 19 PRT Artificial Sequence Synthesized 176 Gly Arg Ser Phe Cys Met Asp Trp Pro Ala His Lys Ser Cys Thr Pro 1 5 10 15 Leu Met Leu 177 15 PRT Artificial Sequence Synthesized 177 Gly Thr Glu Val Cys Tyr Phe Pro Gly Ile Cys Trp Gly Gly Gly 1 5 10 15 178 20 PRT Artificial Sequence Synthesized 178 Gly Val Arg Thr Phe Cys Gln Asp Trp Pro Ala His Asn Ser Cys Lys 1 5 10 15 Leu Leu Arg Gly 20 179 15 PRT Artificial Sequence Synthesized 179 His Ala Glu Ile Cys Tyr Phe Pro Gly Ile Cys Trp Thr Glu Arg 1 5 10 15 180 15 PRT Artificial Sequence Synthesized 180 Ile Val Glu Met Cys Tyr Tyr Pro Gly Ile Cys Trp Ile Ser Pro 1 5 10 15 181 11 PRT Artificial Sequence Synthesized 181 Lys Leu Cys Tyr Phe Pro Gly Ile Cys Trp Ser 1 5 10 182 11 PRT Artificial Sequence Synthesized 182 Lys Thr Cys Tyr Phe Pro Gly Ile Cys Trp Met 1 5 10 183 15 PRT Artificial Sequence Synthesized 183 Lys Thr Glu Ile Cys Tyr Phe Pro Gly Ile Cys Trp Met Ser Gly 1 5 10 15 184 11 PRT Artificial Sequence Synthesized 184 Lys Val Cys Tyr Phe Pro Gly Ile Cys Trp Met 1 5 10 185 15 PRT Artificial Sequence Synthesized 185 Leu Ala Glu Met Cys Tyr Phe Pro Gly Ile Cys Trp Met Ser Ala 1 5 10 15 186 20 PRT Artificial Sequence Synthesized 186 Leu Val Pro Glu Arg Ile Val Cys Tyr Phe Glu Ser Ile Cys Tyr Glu 1 5 10 15 Arg Ser Glu Leu 20 187 20 PRT Artificial Sequence Synthesized 187 Met Glu Leu Trp Cys Asp Ser Thr Leu Met Ala Tyr Asp Leu Cys Gly 1 5 10 15 Asp Phe Asn Met 20 188 20 PRT Artificial Sequence Synthesized 188 Met Glu Pro Ser Arg Ser Val Cys Tyr Ala Glu Gly Ile Cys Phe Asp 1 5 10 15 Arg Gly Glu Gln 20 189 15 PRT Artificial Sequence Synthesized 189 Asn Asp Glu Ile Cys Tyr Phe Pro Gly Val Cys Trp Lys Ser Gly 1 5 10 15 190 8 PRT Artificial Sequence Synthesized 190 Gln Cys Phe Pro Gly Trp Val Lys 1 5 191 15 PRT Artificial Sequence Synthesized 191 Gln Thr Glu Leu Cys Tyr Phe Pro Gly Ile Cys Trp Asn Glu Ser 1 5 10 15 192 20 PRT Artificial Sequence Synthesized 192 Gln Thr Arg Ser Phe Cys Ala Asp Trp Pro Arg His Glu Ser Cys Lys 1 5 10 15 Pro Leu Arg Gly 20 193 20 PRT Artificial Sequence Synthesized 193 Arg Ala Ala Glu Ser Ser Val Cys Tyr Trp Pro Gly Ile Cys Phe Asp 1 5 10 15 Arg Thr Glu Gln 20 194 20 PRT Artificial Sequence Synthesized 194 Arg Ala Pro Glu Arg Trp Val Cys Tyr Trp Glu Gly Ile Cys Phe Asp 1 5 10 15 Arg Tyr Glu Gln 20 195 15 PRT Artificial Sequence Synthesized 195 Arg Asp Thr Val Cys Tyr Phe Pro Gly Ile Cys Trp Met Ala Ser 1 5 10 15 196 20 PRT Artificial Sequence Synthesized 196 Arg Glu Pro Ala Ser Leu Val Cys Tyr Phe Glu Asp Ile Cys Phe Val 1 5 10 15 Arg Ala Glu Ala 20 197 18 PRT Artificial Sequence Synthesized 197 Arg Gly Pro Asp Val Cys Tyr Trp Pro Ser Ile Cys Phe Glu Arg Ser 1 5 10 15 Met Pro 198 20 PRT Artificial Sequence Synthesized 198 Arg Met Pro Ala Ser Leu Pro Cys Tyr Trp Glu Thr Ile Cys Tyr Glu 1 5 10 15 Ser Ser Glu Gln 20 199 15 PRT Artificial Sequence Synthesized 199 Arg Arg Thr Cys Asp Trp Pro His Asn Ser Cys Lys Leu Arg Gly 1 5 10 15 200 20 PRT Artificial Sequence Synthesized 200 Arg Thr Ala Glu Ser Leu Val Cys Tyr Trp Pro Gly Ile Cys Phe Ala 1 5 10 15 Gln Ser Glu Arg 20 201 15 PRT Artificial Sequence Synthesized 201 Ser Gly Ala Ile Cys Tyr Val Pro Gly Ile Cys Trp Thr His Ala 1 5 10 15 202 15 PRT Artificial Sequence Synthesized 202 Ser Arg Glu Val Cys Tyr Tyr Pro Gly Ile Cys Trp Asn Gly Ala 1 5 10 15 203 15 PRT Artificial Sequence Synthesized 203 Ser Tyr Ala Pro Cys Tyr Phe Pro Gly Ile Cys Trp Met Gly Asn 1 5 10 15 204 15 PRT Artificial Sequence Synthesized 204 Thr Thr Glu Met Cys Tyr Phe Pro Gly Ile Cys Trp Lys Thr Glu 1 5 10 15 205 15 PRT Artificial Sequence Synthesized 205 Val Gln Glu Val Cys Tyr Phe Pro Gly Ile Cys Trp Met Gln Glu 1 5 10 15 206 15 PRT Artificial Sequence Synthesized 206 Val Arg Asp Met Cys Tyr Phe Pro Gly Ile Cys Trp Lys Ser Glu 1 5 10 15 207 20 PRT Artificial Sequence Synthesized 207 Asn Arg Gln Met Glu Asp Ile Cys Leu Pro Gln Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Asp Phe 20 208 20 PRT Artificial Sequence Synthesized 208 Gln Arg His Pro Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Asp Asp 20 209 15 PRT Artificial Sequence Synthesized 209 Asp Phe Asp Leu Cys Leu Pro Asp Trp Gly Cys Leu Trp Asp Asp 1 5 10 15 210 11 PRT Artificial Sequence Synthesized 210 Asp Ile Cys Leu Glu Arg Trp Gly Cys Leu Trp 1 5 10 211 11 PRT Artificial Sequence Synthesized 211 Asp Ile Cys Leu Pro Ala Trp Gly Cys Leu Trp 1 5 10 212 11 PRT Artificial Sequence Synthesized 212 Asp Ile Cys Leu Pro Asp Trp Gly Cys Leu Trp 1 5 10 213 11 PRT Artificial Sequence Synthesized 213 Asp Ile Cys Leu Pro Glu Trp Gly Cys Leu Trp 1 5 10 214 11 PRT Artificial Sequence Synthesized 214 Asp Ile Cys Leu Pro Val Trp Gly Cys Leu Trp 1 5 10 215 11 PRT Artificial Sequence Synthesized 215 Asp Leu Cys Leu Pro Glu Trp Gly Cys Leu Trp 1 5 10 216 11 PRT Artificial Sequence Synthesized 216 Asp Leu Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 217 11 PRT Artificial Sequence Synthesized 217 Asp Leu Cys Leu Pro Val Trp Gly Cys Leu Trp 1 5 10 218 15 PRT Artificial Sequence Synthesized 218 Asp Ser Cys Gly Asp Leu Leu Arg Leu Gly Leu Glu Cys Trp Ala 1 5 10 15 219 15 PRT Artificial Sequence Synthesized 219 Asp Thr Cys Ala Asp Leu Val Arg Leu Gly Leu Glu Cys Trp Ala 1 5 10 15 220 15 PRT Artificial Sequence Synthesized 220 Asp Thr Cys Asp Asp Leu Val Gln Leu Gly Leu Glu Cys Trp Ala 1 5 10 15 221 15 PRT Artificial Sequence Synthesized 221 Asp Thr Cys Glu Asp Leu Val Arg Leu Gly Leu Glu Cys Trp Ala 1 5 10 15 222 15 PRT Artificial Sequence Synthesized 222 Asp Thr Cys Ser Asp Leu Val Gly Leu Gly Leu Glu Cys Trp Ala 1 5 10 15 223 15 PRT Artificial Sequence Synthesized 223 Glu Glu Asp Leu Cys Leu Pro Val Trp Gly Cys Leu Trp Gly Ala 1 5 10 15 224 15 PRT Artificial Sequence Synthesized 224 Glu Glu Asp Val Cys Leu Pro Val Trp Gly Cys Leu Trp Glu Gly 1 5 10 15 225 15 PRT Artificial Sequence Synthesized 225 Glu Phe Asp Leu Cys Leu Pro Thr Trp Gly Cys Leu Trp Glu Asp 1 5 10 15 226 20 PRT Artificial Sequence Synthesized 226 Glu Arg Gln Met Glu Asp Phe Cys Leu Pro Gln Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Gly Val 20 227 20 PRT Artificial Sequence Synthesized 227 Glu Arg Gln Met Val Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Gly Phe 20 228 15 PRT Artificial Sequence Synthesized 228 Glu Trp Asp Val Cys Phe Pro Ala Trp Gly Cys Leu Trp Asp Gln 1 5 10 15 229 15 PRT Artificial Sequence Synthesized 229 Glu Trp Asp Val Cys Leu Pro His Trp Gly Cys Leu Trp Asp Gly 1 5 10 15 230 15 PRT Artificial Sequence Synthesized 230 Phe Glu Asp Phe Cys Leu Pro Asn Trp Gly Cys Leu Trp Gly Ser 1 5 10 15 231 15 PRT Artificial Sequence Synthesized 231 Phe Glu Asp Leu Cys Val Val Arg Trp Gly Cys Leu Trp Gly Asp 1 5 10 15 232 20 PRT Artificial Sequence Synthesized 232 Gly Arg Gln Val Val Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Glu Glu Gly Leu 20 233 20 PRT Artificial Sequence Synthesized 233 Lys Met Gly Arg Val Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Glu Leu 20 234 20 PRT Artificial Sequence Synthesized 234 Lys Ser Arg Met Gly Asp Phe Cys Leu Pro Glu Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Glu Leu 20 235 20 PRT Artificial Sequence Synthesized 235 Leu Arg Ile Phe Glu Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Gly Glu Gly Phe 20 236 15 PRT Artificial Sequence Synthesized 236 Met Asp Asp Ile Cys Leu His His Trp Gly Cys Leu Trp Asp Glu 1 5 10 15 237 15 PRT Artificial Sequence Synthesized 237 Met Asp Asp Leu Cys Leu Pro Asn Trp Gly Cys Leu Trp Gly Asp 1 5 10 15 238 15 PRT Artificial Sequence Synthesized 238 Met Phe Asp Leu Cys Leu Pro Lys Trp Gly Cys Leu Trp Gly Asn 1 5 10 15 239 15 PRT Artificial Sequence Synthesized 239 Met Trp Asp Val Cys Phe Pro Asp Trp Gly Cys Leu Trp Asp Val 1 5 10 15 240 15 PRT Artificial Sequence Synthesized 240 Asn Thr Cys Ala Asp Leu Val Arg Leu Gly Leu Glu Cys Trp Ala 1 5 10 15 241 15 PRT Artificial Sequence Synthesized 241 Asn Trp Asp Leu Cys Phe Pro Asp Trp Gly Cys Leu Trp Asp Asp 1 5 10 15 242 20 PRT Artificial Sequence Synthesized 242 Gln Gly Asp Phe Trp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Ser 1 5 10 15 Gly Glu Gly Tyr 20 243 20 PRT Artificial Sequence Synthesized 243 Gln Gly Gly Met Gly Asp Phe Cys Leu Pro Gln Trp Gly Cys Leu Trp 1 5 10 15 Gly Glu Asp Leu 20 244 20 PRT Artificial Sequence Synthesized 244 Gln Gly Tyr Met Val Asp Phe Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Ala Asn 20 245 20 PRT Artificial Sequence Synthesized 245 Gln Met His Met Met Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Thr Ser 20 246 20 PRT Artificial Sequence Synthesized 246 Gln Met Gln Met Ser Asp Phe Cys Leu Pro Gln Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Gly Tyr 20 247 20 PRT Artificial Sequence Synthesized 247 Gln Arg His Met Met Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Gly Tyr 20 248 20 PRT Artificial Sequence Synthesized 248 Gln Arg Leu Met Trp Glu Ile Cys Leu Pro Leu Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Gly Leu 20 249 20 PRT Artificial Sequence Synthesized 249 Gln Arg Pro Ile Met Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Glu Asp Gly Phe 20 250 20 PRT Artificial Sequence Synthesized 250 Gln Arg Gln Ile Met Asp Phe Cys Leu Pro His Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Gly Phe 20 251 20 PRT Artificial Sequence Synthesized 251 Gln Arg Gln Val Val Asp Phe Cys Leu Pro Gln Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Gly Ser 20 252 20 PRT Artificial Sequence Synthesized 252 Gln Ser Gln Leu Glu Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Gly Phe 20 253 20 PRT Artificial Sequence Synthesized 253 Gln Ser Tyr Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Ser 1 5 10 15 Asp Asp Ala Ser 20 254 20 PRT Artificial Sequence Synthesized 254 Arg Trp Gln Thr Glu Asp Val Cys Leu Pro Lys Trp Gly Cys Leu Phe 1 5 10 15 Gly Asp Gly Val 20 255 15 PRT Artificial Sequence Synthesized 255 Ser Glu Asp Phe Cys Leu Pro Val Trp Gly Cys Leu Trp Glu Asp 1 5 10 15 256 15 PRT Artificial Sequence Synthesized 256 Val Trp Asp Leu Cys Leu Pro Gln Trp Gly Cys Leu Trp Asp Glu 1 5 10 15 257 15 PRT Artificial Sequence Synthesized 257 Trp Asp Asp Ile Cys Phe Arg Asp Trp Gly Cys Leu Trp Gly Ser 1 5 10 15 258 15 PRT Artificial Sequence Synthesized 258 Trp Glu Asp Leu Cys Leu Pro Asp Trp Gly Cys Leu Trp Glu Asp 1 5 10 15 259 20 PRT Artificial Sequence Synthesized 259 His Arg Leu Val Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Asn Asp Phe 20 260 20 PRT Artificial Sequence Synthesized 260 His Ser Gln Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Glu Leu 20 261 20 PRT Artificial Sequence Synthesized 261 Leu Ile Phe Met Glu Asp Val Cys Leu Pro Gln Trp Gly Cys Leu Trp 1 5 10 15 Glu Asp Gly Val 20 262 20 PRT Artificial Sequence Synthesized 262 Leu Arg Leu Met Asp Asn Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Asp Asp Gly Phe 20 263 20 PRT Artificial Sequence Synthesized 263 Leu Trp Ala Met Glu Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Glu Asp Asp Phe 20 264 20 PRT Artificial Sequence Synthesized 264 Gln Arg Asp Met Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Asp Gly Val 20 265 20 PRT Artificial Sequence Synthesized 265 Gln Arg Leu Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Arg Phe 20 266 20 PRT Artificial Sequence Synthesized 266 Gln Trp His Met Glu Asp Ile Cys Leu Pro Gln Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Val Leu 20 267 20 PRT Artificial Sequence Synthesized 267 Gln Trp Gln Met Glu Asn Val Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Glu Glu Leu Asp 20 268 20 PRT Artificial Sequence Synthesized 268 Gln Trp Gln Val Met Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Ala Asp Glu Tyr 20 269 11 PRT Artificial Sequence Synthesized 269 Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 270 20 PRT Artificial Sequence Synthesized 270 Ala Ala Gln Val Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Ser Glu Tyr Ala 20 271 20 PRT Artificial Sequence Synthesized 271 Ala Gly Trp Ala Ala Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Glu Asp Val 20 272 18 PRT Artificial Sequence Synthesized 272 Ala Leu Phe Glu Asp Val Cys Leu Pro Val Trp Gly Cys Leu Trp Gly 1 5 10 15 Gly Glu 273 20 PRT Artificial Sequence Synthesized 273 Ala Gln Ala Met Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Ala Glu Ile 20 274 20 PRT Artificial Sequence Synthesized 274 Ala Ser Asp Pro Gly Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Glu Ser Phe 20 275 20 PRT Artificial Sequence Synthesized 275 Ala Ser Asp Arg Gly Asp Leu Cys Leu Pro Tyr Trp Gly Cys Leu Trp 1 5 10 15 Gly Pro Asp Gly 20 276 18 PRT Artificial Sequence Synthesized 276 Ala Ser Glu Trp Asp Val Cys Leu Pro Thr Trp Gly Cys Leu Trp Met 1 5 10 15 Glu Gly 277 20 PRT Artificial Sequence Synthesized 277 Ala Ser Asn Trp Glu Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Glu Arg Asn 20 278 20 PRT Artificial Sequence Synthesized 278 Ala Ser Thr Pro Arg Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Ser Glu Asp Ala 20 279 20 PRT Artificial Sequence Synthesized 279 Ala Ser Val Val Asp Asp Ile Cys Leu Pro Val Trp Gly Cys Leu Trp 1 5 10 15 Gly Glu Asp Ile 20 280 20 PRT Artificial Sequence Synthesized 280 Ala Thr Met Glu Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Ala Glu Glu 20 281 18 PRT Artificial Sequence Synthesized 281 Ala Tyr Ser Ala Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Met 1 5 10 15 Ser Glu 282 20 PRT Artificial Sequence Synthesized 282 Asp Glu Asp Phe Glu Asp Tyr Cys Leu Pro Pro Trp Gly Cys Leu Trp 1 5 10 15 Gly Ser Ser Met 20 283 20 PRT Artificial Sequence Synthesized 283 Asp Gly Glu Glu Gly Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Ala Leu Glu His 20 284 18 PRT Artificial Sequence Synthesized 284 Glu Asp Trp Glu Asp Ile Cys Leu Pro Gln Trp Gly Cys Leu Trp Glu 1 5 10 15 Gly Met 285 18 PRT Artificial Sequence Synthesized 285 Glu Asp Trp Thr Asp Leu Cys Leu Pro Ala Trp Gly Cys Leu Trp Asp 1 5 10 15 Thr Glu 286 18 PRT Artificial Sequence Synthesized 286 Glu Glu Asp Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asn 1 5 10 15 Thr Ser 287 20 PRT Artificial Sequence Synthesized 287 Glu Gly Glu Glu Val Asp Ile Cys Leu Pro Gln Trp Gly Cys Leu Trp 1 5 10 15 Gly Tyr Pro Val 20 288 20 PRT Artificial Sequence Synthesized 288 Glu Gly Thr Trp Asp Asp Phe Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Leu Gly Glu Arg 20 289 18 PRT Artificial Sequence Synthesized 289 Glu Gly Tyr Trp Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Leu Glu 290 18 PRT Artificial Sequence Synthesized 290 Glu Leu Gly Glu Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Ser Glu 291 20 PRT Artificial Sequence Synthesized 291 Glu Arg Trp Glu Gly Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Glu Ser Gly 20 292 18 PRT Artificial Sequence Synthesized 292 Glu Thr Trp Ser Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Ala Ser 293 20 PRT Artificial Sequence Synthesized 293 Glu Val Gly Asp Leu Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Asn Asp Lys 20 294 20 PRT Artificial Sequence Synthesized 294 Phe Arg Asp Gly Glu Asp Phe Cys Leu Pro Gln Trp Gly Cys Leu Trp 1 5 10 15 Ala Asp Thr Ser 20 295 20 PRT Artificial Sequence Synthesized 295 Gly Asp Met Val Asn Asp Phe Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Ser Glu Asn 20 296 20 PRT Artificial Sequence Synthesized 296 Gly Asp Trp Met His Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Asp Glu Lys Ala 20 297 18 PRT Artificial Sequence Synthesized 297 Gly Asp Tyr Val Asp Leu Cys Leu Pro Gly Trp Gly Cys Leu Trp Glu 1 5 10 15 Asp Gly 298 20 PRT Artificial Sequence Synthesized 298 Gly Ile Glu Trp Gly Asp Thr Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Arg Val Glu Gly 20 299 20 PRT Artificial Sequence Synthesized 299 Gly Gln Gln Gly Glu Asp Val Cys Leu Pro Val Trp Gly Cys Leu Trp 1 5 10 15 Asp Thr Ser Ser 20 300 20 PRT Artificial Sequence Synthesized 300 Gly Arg Met Gly Thr Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Glu Val Glu 20 301 20 PRT Artificial Sequence Synthesized 301 Gly Arg Tyr Pro Met Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Asp Ser Ala 20 302 20 PRT Artificial Sequence Synthesized 302 Gly Ser Ala Gly Asp Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Arg Gly Ala 20 303 18 PRT Artificial Sequence Synthesized 303 Gly Val Leu Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Pro Lys 304 20 PRT Artificial Sequence Synthesized 304 His Ala Ser Asp Trp Asp Val Cys Leu Pro Gly Trp Gly Cys Leu Trp 1 5 10 15 Glu Glu Asp Asp 20 305 20 PRT Artificial Sequence Synthesized 305 His Glu Trp Glu Arg Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Arg Asp Gly Asp 20 306 18 PRT Artificial Sequence Synthesized 306 His Met Met Asp Asp Val Cys Leu Pro Gly Trp Gly Cys Leu Trp Ala 1 5 10 15 Ser Glu 307 18 PRT Artificial Sequence Synthesized 307 Ile Asp Tyr Thr Asp Leu Cys Leu Pro Ala Trp Gly Cys Leu Trp Glu 1 5 10 15 Leu Glu 308 18 PRT Artificial Sequence Synthesized 308 Ile Glu His Glu Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp Ala 1 5 10 15 Val Asp 309 18 PRT Artificial Sequence Synthesized 309 Ile Ser Glu Trp Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp 1 5 10 15 Arg Ser 310 18 PRT Artificial Sequence Synthesized 310 Ile Ser Trp Ala Asp Val Cys Leu Pro Lys Trp Gly Cys Leu Trp Gly 1 5 10 15 Lys Asp 311 18 PRT Artificial Sequence Synthesized 311 Ile Ser Trp Gly Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Gly Ser 312 20 PRT Artificial Sequence Synthesized 312 Lys Lys Val Ser Gly Asp Ile Cys Leu Pro Ile Trp Gly Cys Leu Trp 1 5 10 15 Asp Asn Asp Tyr 20 313 18 PRT Artificial Sequence Synthesized 313 Lys Leu Trp Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Ser 1 5 10 15 Pro Leu 314 18 PRT Artificial Sequence Synthesized 314 Leu Ala Trp Pro Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Gly Met 315 20 PRT Artificial Sequence Synthesized 315 Leu Gly Val Thr His Asp Thr Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Asp Glu Val Gly 20 316 20 PRT Artificial Sequence Synthesized 316 Leu Leu Glu Ser Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 His Glu Asp Gly 20 317 18 PRT Artificial Sequence Synthesized 317 Leu Asn Glu Ser Asp Ile Cys Leu Pro Thr Trp Gly Cys Leu Trp Gly 1 5 10 15 Val Asp 318 18 PRT Artificial Sequence Synthesized 318 Leu Pro Glu Gln Asp Val Cys Leu Pro Val Trp Gly Cys Leu Trp Asp 1 5 10 15 Ala Asn 319 20 PRT Artificial Sequence Synthesized 319 Leu Val Trp Glu Glu Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Gly Ala Glu Asp 20 320 18 PRT Artificial Sequence Synthesized 320 Met Ala Trp Gly Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp Ala 1 5 10 15 Gly Gly 321 20 PRT Artificial Sequence Synthesized 321 Met Gln Ala Glu Ser Asp Phe Cys Leu Pro His Trp Gly Cys Leu Trp 1 5 10 15 Asp Glu Gly Thr 20 322 20 PRT Artificial Sequence Synthesized 322 Met Gln Gly Pro Leu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Gly Val Asp 20 323 18 PRT Artificial Sequence Synthesized 323 Asn Glu Glu Trp Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Gly Val 324 20 PRT Artificial Sequence Synthesized 324 Asn Val Gly Trp Asn Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Ala Gln Glu Ser 20 325 18 PRT Artificial Sequence Synthesized 325 Gln Glu Leu Gln Asp Phe Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Val Gly 326 20 PRT Artificial Sequence Synthesized 326 Gln Gly Val Glu Trp Asp Val Cys Leu Pro Gln Trp Gly Cys Leu Trp 1 5 10 15 Thr Arg Glu Val 20 327 20 PRT Artificial Sequence Synthesized 327 Gln Met Pro Leu Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Gly Arg Glu 20 328 18 PRT Artificial Sequence Synthesized 328 Gln Arg Glu Trp Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp Ser 1 5 10 15 Asp Val 329 18 PRT Artificial Sequence Synthesized 329 Gln Arg Phe Trp Asp Thr Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Gly Asp 330 20 PRT Artificial Sequence Synthesized 330 Arg Glu Glu Trp Gly Asp Leu Cys Leu Pro Thr Trp Gly Cys Leu Trp 1 5 10 15 Glu Thr Lys Lys 20 331 20 PRT Artificial Sequence Synthesized 331 Arg Leu Asp Ala Trp Asp Ile Cys Leu Pro Gln Trp Gly Cys Leu Trp 1 5 10 15 Glu Glu Pro Ser 20 332 18 PRT Artificial Sequence Synthesized 332 Arg Val Phe Thr Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp 1 5 10 15 Leu Gly 333 20 PRT Artificial Sequence Synthesized 333 Arg Val Trp Thr Glu Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Ser Glu Gly Asn 20 334 20 PRT Artificial Sequence Synthesized 334 Ser Glu Ala Pro Gly Asp Tyr Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Ala Gln Glu Lys 20 335 18 PRT Artificial Sequence Synthesized 335 Ser Gly Trp Asp Asp Val Cys Leu Pro Val Trp Gly Cys Leu Trp Gly 1 5 10 15 Pro Ser 336 20 PRT Artificial Sequence Synthesized 336 Ser Ile Arg Glu Tyr Asp Val Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Glu Pro Ser Ala 20 337 20 PRT Artificial Sequence Synthesized 337 Ser Pro Thr Glu Trp Asp Met Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Ala Leu 20 338 18 PRT Artificial Sequence Synthesized 338 Ser Ser Ala Ser Asp Tyr Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Asp Leu 339 20 PRT Artificial Sequence Synthesized 339 Ser Ser Gly Leu Glu Asp Ile Cys Leu Pro Asn Trp Gly Cys Leu Trp 1 5 10 15 Ala Asp Gly Ser 20 340 20 PRT Artificial Sequence Synthesized 340 Ser Val Gly Trp Gly Asp Ile Cys Leu Pro Val Trp Gly Cys Leu Trp 1 5 10 15 Gly Glu Gly Gly 20 341 18 PRT Artificial Sequence Synthesized 341 Ser Trp Gln Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Val Asp 342 18 PRT Artificial Sequence Synthesized 342 Ser Tyr Glu Thr Asp Val Cys Leu Pro Tyr Trp Gly Cys Leu Trp Glu 1 5 10 15 Asp Ala 343 18 PRT Artificial Sequence Synthesized 343 Ser Tyr Trp Gly Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp Ser 1 5 10 15 Glu Ala 344 20 PRT Artificial Sequence Synthesized 344 Thr Ala Met Asp Glu Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Ser Gly Ser 20 345 20 PRT Artificial Sequence Synthesized 345 Thr Glu Glu Asn Trp Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Asp Trp 20 346 20 PRT Artificial Sequence Synthesized 346 Thr Glu Ile Gly Gln Asp Phe Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Val Pro Gly Thr 20 347 18 PRT Artificial Sequence Synthesized 347 Thr Leu Glu Trp Asp Met Cys Leu Pro Arg Trp Gly Cys Leu Trp Thr 1 5 10 15 Glu Gln 348 20 PRT Artificial Sequence Synthesized 348 Thr Leu Gly Trp Pro Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Arg Glu Ser Asp 20 349 20 PRT Artificial Sequence Synthesized 349 Thr Leu Ser Asn Gln Asp Ile Cys Leu Pro Gly Trp Gly Cys Leu Trp 1 5 10 15 Gly Gly Ile Asn 20 350 20 PRT Artificial Sequence Synthesized 350 Thr Ser Gly Ser Asp Asp Ile Cys Leu Pro Val Trp Gly Cys Leu Trp 1 5 10 15 Gly Glu Asp Ser 20 351 20 PRT Artificial Sequence Synthesized 351 Thr Ser Thr Gly Gly Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Asp Ser Ser Glu 20 352 19 PRT Artificial Sequence Synthesized 352 Thr Trp Pro Gly Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Ala Glu Ser 353 18 PRT Artificial Sequence Synthesized 353 Val Gly Glu Phe Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp 1 5 10 15 Ala Glu 354 20 PRT Artificial Sequence Synthesized 354 Val Ser Glu Met Asp Asp Ile Cys Leu Pro Leu Trp Gly Cys Leu Trp 1 5 10 15 Ala Asp Ala Pro 20 355 20 PRT Artificial Sequence Synthesized 355 Val Ser Glu Trp Glu Asp Ile Cys Leu Pro Ser Trp Gly Cys Leu Trp 1 5 10 15 Glu Thr Gln Asp 20 356 18 PRT Artificial Sequence Synthesized 356 Val Thr Ser Trp Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Glu Asp 357 20 PRT Artificial Sequence Synthesized 357 Val Val Gly Asp Gly Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Asp Gln Ala Arg 20 358 20 PRT Artificial Sequence Synthesized 358 Val Val Trp Asp Asp Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Glu Tyr Gly 20 359 20 PRT Artificial Sequence Synthesized 359 Trp Asp His Glu Leu Asp Phe Cys Leu Pro Val Trp Gly Cys Leu Trp 1 5 10 15 Ala Glu Asp Val 20 360 18 PRT Artificial Sequence Synthesized 360 Trp Leu Trp Glu Asp Leu Cys Leu Pro Lys Trp Gly Cys Leu Trp Glu 1 5 10 15 Glu Asp 361 20 PRT Artificial Sequence Synthesized 361 Trp Ser Asp Ser Asp Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Asn Val Ala 20 362 20 PRT Artificial Sequence Synthesized 362 Trp Thr Glu Ser Glu Asp Ile Cys Leu Pro Gly Trp Gly Cys Leu Trp 1 5 10 15 Gly Pro Glu Val 20 363 20 PRT Artificial Sequence Synthesized 363 Trp Val Glu Glu Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Ser Val Glu 20 364 20 PRT Artificial Sequence Synthesized 364 Trp Val Pro Phe Glu Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Ser Ser Tyr Gln 20 365 18 PRT Artificial Sequence Synthesized 365 Xaa Xaa Xaa Xaa Asp Xaa Cys Leu Pro Xaa Trp Gly Cys Leu Trp Xaa 1 5 10 15 Xaa Xaa 366 20 PRT Artificial Sequence Synthesized 366 Xaa Xaa Xaa Xaa Xaa Asp Xaa Cys Leu Pro Xaa Trp Gly Cys Leu Trp 1 5 10 15 Xaa Xaa Xaa Xaa 20 367 18 PRT Artificial Sequence Synthesized 367 Ala Phe Trp Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Glu Asp 368 20 PRT Artificial Sequence Synthesized 368 Ala Gly Leu Asp Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Lys Glu Ala 20 369 20 PRT Artificial Sequence Synthesized 369 Ala Gly Met Met Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gln Gly Glu Pro 20 370 20 PRT Artificial Sequence Synthesized 370 Ala Pro Gly Asp Trp Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Asp Asp Asp Ala 20 371 20 PRT Artificial Sequence Synthesized 371 Ala Gln Leu Phe Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Ser Asp Gly Tyr 20 372 20 PRT Artificial Sequence Synthesized 372 Ala Arg Thr Met Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Ala Ser Asp 20 373 20 PRT Artificial Sequence Synthesized 373 Ala Val Ser Trp Ala Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Arg Ala Asp 20 374 20 PRT Artificial Sequence Synthesized 374 Ala Trp Leu Asp Glu Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Asn Thr Gly Val 20 375 20 PRT Artificial Sequence Synthesized 375 Ala Trp Gln Asp Phe Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Pro Glu Ser 20 376 20 PRT Artificial Sequence Synthesized 376 Asp Thr Thr Trp Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Ser Glu Glu Ala 20 377 18 PRT Artificial Sequence Synthesized 377 Asp Trp Gly Arg Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp 1 5 10 15 Glu Glu 378 18 PRT Artificial Sequence Synthesized 378 Glu Ala Trp Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Leu Glu 379 20 PRT Artificial Sequence Synthesized 379 Glu Gly Phe Leu Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly His Gln Ala 20 380 20 PRT Artificial Sequence Synthesized 380 Glu Gln Trp Leu His Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Asp Asp Thr Asp 20 381 20 PRT Artificial Sequence Synthesized 381 Glu Thr Gly Trp Pro Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Glu Gly Glu 20 382 20 PRT Artificial Sequence Synthesized 382 Phe Glu Leu Gly Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Glu His Asn 20 383 18 PRT Artificial Sequence Synthesized 383 Phe Ile Thr Gln Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Glu Asn 384 18 PRT Artificial Sequence Synthesized 384 Phe Leu Trp Arg Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Ser 1 5 10 15 Glu Gly 385 20 PRT Artificial Sequence Synthesized 385 Phe Ser Leu Asp Glu Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Gly Ala Glu Lys 20 386 18 PRT Artificial Sequence Synthesized 386 Phe Val His Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Glu Gly 387 20 PRT Artificial Sequence Synthesized 387 Gly Ala Ser Leu Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Pro Glu Asp 20 388 20 PRT Artificial Sequence Synthesized 388 Gly Asp Leu Gly Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Asp Glu Tyr Pro 20 389 20 PRT Artificial Sequence Synthesized 389 Gly Glu Gly Trp Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Ala Glu Asp Glu 20 390 20 PRT Artificial Sequence Synthesized 390 Gly Glu Trp Trp Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Ser Ser Ser 20 391 18 PRT Artificial Sequence Synthesized 391 Gly Leu Gly Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Arg Asp 392 20 PRT Artificial Sequence Synthesized 392 Gly Leu Met Gly Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Lys Gly Asp Ile 20 393 18 PRT Artificial Sequence Synthesized 393 Gly Met Phe Asp Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp Gly 1 5 10 15 Leu Gly 394 18 PRT Artificial Sequence Synthesized 394 Gly Pro Gly Trp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Glu Glu 395 18 PRT Artificial Sequence Synthesized 395 Gly Pro Trp Tyr Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp 1 5 10 15 Gly Val 396 20 PRT Artificial Sequence Synthesized 396 Gly Ser Leu Glu Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Ile Asp Glu 20 397 18 PRT Artificial Sequence Synthesized 397 Gly Trp Asp Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Asp Gly 398 20 PRT Artificial Sequence Synthesized 398 Gly Trp His Asp Arg Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Gln Asn Asp 20 399 20 PRT Artificial Sequence Synthesized 399 Gly Trp Leu Glu Glu Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Gly Ala Asp Asn 20 400 20 PRT Artificial Sequence Synthesized 400 His Glu Gln Trp Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Gly Ser Tyr 20 401 18 PRT Artificial Sequence Synthesized 401 Leu Glu Tyr Glu Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp Gly 1 5 10 15 Gly Glu 402 18 PRT Artificial Sequence Synthesized 402 Leu Ile Leu Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp 1 5 10 15 Asp Thr 403 18 PRT Artificial Sequence Synthesized 403 Leu Lys Leu Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Glu Ser 404 18 PRT Artificial Sequence Synthesized 404 Leu Leu Asp Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Val Arg 405 20 PRT Artificial Sequence Synthesized 405 Leu Leu Gly Gly His Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Gly Asp Val 20 406 18 PRT Artificial Sequence Synthesized 406 Leu Leu Thr Arg Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp Gly 1 5 10 15 Ser Asp 407 18 PRT Artificial Sequence Synthesized 407 Leu Met Ser Pro Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp Glu 1 5 10 15 Gly Asp 408 18 PRT Artificial Sequence Synthesized 408 Leu Arg Trp Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Glu Thr 409 18 PRT Artificial Sequence Synthesized 409 Leu Val Leu Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Ser Asp 410 18 PRT Artificial Sequence Synthesized 410 Leu Tyr Leu Arg Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp Glu 1 5 10 15 Ala Asp 411 18 PRT Artificial Sequence Synthesized 411 Met Leu Ser Arg Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Glu Glu 412 18 PRT Artificial Sequence Synthesized 412 Met Pro Trp Thr Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Ser 1 5 10 15 Glu Ser 413 20 PRT Artificial Sequence Synthesized 413 Met Arg Trp Ser Ser Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Glu Glu 20 414 18 PRT Artificial Sequence Synthesized 414 Asn Trp Tyr Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp 1 5 10 15 Val Glu 415 17 PRT Artificial Sequence Synthesized 415 Gln Asp Trp Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Asp 416 20 PRT Artificial Sequence Synthesized 416 Gln Phe Glu Trp Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Val Glu Val 20 417 20 PRT Artificial Sequence Synthesized 417 Gln Gly Trp Trp His Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Glu Gly Glu 20 418 20 PRT Artificial Sequence Synthesized 418 Gln Arg Val Asp Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Glu Asn Ser 20 419 18 PRT Artificial Sequence Synthesized 419 Gln Ser Trp Pro Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp Gly 1 5 10 15 Glu Gly 420 20 PRT Artificial Sequence Synthesized 420 Arg Glu Gly Trp Pro Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Ser Glu Thr Gly 20 421 20 PRT Artificial Sequence Synthesized 421 Arg Glu Leu Trp Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu His Ala Thr 20 422 20 PRT Artificial Sequence Synthesized 422 Arg Leu Glu Leu Met Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Asp Pro Gln Asp 20 423 18 PRT Artificial Sequence Synthesized 423 Arg Leu Gly Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp 1 5 10 15 Tyr Gln 424 18 PRT Artificial Sequence Synthesized 424 Arg Leu Gly Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Ala Gly 425 20 PRT Artificial Sequence Synthesized 425 Ser Gly Val Leu Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Glu Ala Gly 20 426 20 PRT Artificial Sequence Synthesized 426 Ser Leu Gly Leu Thr Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Glu Glu Gln 20 427 18 PRT Artificial Sequence Synthesized 427 Ser Pro Trp Met Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Ser Gly 428 20 PRT Artificial Sequence Synthesized 428 Ser Ser Leu Glu Gln Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Gln Asp Ala 20 429 18 PRT Artificial Sequence Synthesized 429 Ser Thr Phe Thr Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Leu Glu 430 20 PRT Artificial Sequence Synthesized 430 Ser Val Gly Trp Gly Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Ala Glu Ser Asp 20 431 20 PRT Artificial Sequence Synthesized 431 Ser Val Leu Ser Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Trp Asp Phe Ser 20 432 18 PRT Artificial Sequence Synthesized 432 Ser Val Leu Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Glu Ser 433 18 PRT Artificial Sequence Synthesized 433 Thr Leu Leu Gln Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Ser Asp 434 20 PRT Artificial Sequence Synthesized 434 Thr Leu Met Ser Asn Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Asp Glu Pro Lys 20 435 20 PRT Artificial Sequence Synthesized 435 Thr Leu Val Leu Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Asp Met Thr Asp 20 436 20 PRT Artificial Sequence Synthesized 436 Thr Ser Leu Ala Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Ser Glu Asp Gly 20 437 20 PRT Artificial Sequence Synthesized 437 Thr Ser Leu Leu Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Tyr Glu Glu Gly 20 438 18 PRT Artificial Sequence Synthesized 438 Thr Trp Phe Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Pro Gly 439 20 PRT Artificial Sequence Synthesized 439 Thr Trp Gln Gly Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Asp Thr Glu Val 20 440 20 PRT Artificial Sequence Synthesized 440 Val Glu Met Trp His Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Asp Ser Asn Ala 20 441 20 PRT Artificial Sequence Synthesized 441 Val Gly Val Phe Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Gln Pro Val 20 442 18 PRT Artificial Sequence Synthesized 442 Val His Gln Ala Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Asp Thr 443 18 PRT Artificial Sequence Synthesized 443 Val Leu Leu Gly Asp Ile Cys Leu Pro Leu Trp Gly Cys Leu Trp Gly 1 5 10 15 Glu Asp 444 18 PRT Artificial Sequence Synthesized 444 Val Asn Trp Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Glu Ser 445 20 PRT Artificial Sequence Synthesized 445 Val Pro Ala Met Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Ala Arg Asn 20 446 18 PRT Artificial Sequence Synthesized 446 Val Arg Leu Met Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Glu Glu 447 18 PRT Artificial Sequence Synthesized 447 Val Arg Trp Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Glu Glu 448 20 PRT Artificial Sequence Synthesized 448 Val Ser Leu Gly Asp Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp 1 5 10 15 Glu Pro Glu Ala 20 449 18 PRT Artificial Sequence Synthesized 449 Val Val Trp Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp 1 5 10 15 Lys Glu 450 20 PRT Artificial Sequence Synthesized 450 Val Trp Ile Asp Arg Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Asp Thr Glu Asn 20 451 18 PRT Artificial Sequence Synthesized 451 Val Trp Tyr Lys Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Ala Glu 452 20 PRT Artificial Sequence Synthesized 452 Trp Asp Leu Ala Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Glu Glu Ala 20 453 18 PRT Artificial Sequence Synthesized 453 Trp Asp Val Ala Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Ala 1 5 10 15 Glu Asp 454 18 PRT Artificial Sequence Synthesized 454 Trp Asp Tyr Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Glu Gly 455 18 PRT Artificial Sequence Synthesized 455 Trp Glu Val Gln Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Asp Asp 456 18 PRT Artificial Sequence Synthesized 456 Trp His Met Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Ser 1 5 10 15 Glu Val 457 18 PRT Artificial Sequence Synthesized 457 Trp Lys Asp Phe Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp 1 5 10 15 Asp His 458 18 PRT Artificial Sequence Synthesized 458 Trp Leu Ser Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp 1 5 10 15 Asp Leu 459 18 PRT Artificial Sequence Synthesized 459 Trp Leu Ser Glu Asp Ile Cys Leu Pro Gln Trp Gly Cys Leu Trp Glu 1 5 10 15 Glu Ser 460 18 PRT Artificial Sequence Synthesized 460 Trp Leu Ser Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Ala 1 5 10 15 Ala Asp 461 20 PRT Artificial Sequence Synthesized 461 Trp Arg Trp Asn Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Glu Glu Ala 20 462 18 PRT Artificial Sequence Synthesized 462 Tyr Ile Trp Arg Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Gly Glu 463 18 PRT Artificial Sequence Synthesized 463 Tyr Arg Asp Tyr Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp 1 5 10 15 Glu Arg 464 18 PRT Artificial Sequence Synthesized 464 Ala Gly Glu Trp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp 1 5 10 15 Val Glu 465 18 PRT Artificial Sequence Synthesized 465 Glu Ile Arg Trp Asp Phe Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp 1 5 10 15 Glu Asp 466 18 PRT Artificial Sequence Synthesized 466 Glu Ser Leu Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Ser Gly 467 20 PRT Artificial Sequence Synthesized 467 Glu Val Arg Glu Trp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Asn Trp Arg 20 468 18 PRT Artificial Sequence Synthesized 468 Glu Tyr Trp Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp 1 5 10 15 Trp Gln 469 20 PRT Artificial Sequence Synthesized 469 Phe Gly Gln Glu Trp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Asn Glu Gln 20 470 20 PRT Artificial Sequence Synthesized 470 Ile Trp Gln Leu Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Asp Gly Leu 20 471 18 PRT Artificial Sequence Synthesized 471 Lys Met Trp Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Glu Glu 472 18 PRT Artificial Sequence Synthesized 472 Met Gly Thr Lys Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Ala 1 5 10 15 Glu Ala 473 18 PRT Artificial Sequence Synthesized 473 Met His Glu Trp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Ser Ser 474 20 PRT Artificial Sequence Synthesized 474 Asn Thr Pro Thr Tyr Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Val Pro 20 475 20 PRT Artificial Sequence Synthesized 475 Asn Trp Ala Glu Asn Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Glu Asn 20 476 20 PRT Artificial Sequence Synthesized 476 Gln Pro Val Trp Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Glu Asp His 20 477 18 PRT Artificial Sequence Synthesized 477 Arg Gly Leu His Asp Ala Cys Leu Pro Trp Trp Gly Cys Leu Trp Ala 1 5 10 15 Gly Ser 478 18 PRT Artificial Sequence Synthesized 478 Arg Leu Phe Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gln 1 5 10 15 Gly Glu 479 20 PRT Artificial Sequence Synthesized 479 Ser Ala Arg Glu Trp Asp Ile Cys Leu Pro Thr Trp Gly Cys Leu Trp 1 5 10 15 Glu Lys Asp Ile 20 480 18 PRT Artificial Sequence Synthesized 480 Ser Gly Glu Trp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Glu Gly 481 18 PRT Artificial Sequence Synthesized 481 Ser Met Phe Phe Asp His Cys Leu Pro Met Trp Gly Cys Leu Trp Ala 1 5 10 15 Glu Gln 482 19 PRT Artificial Sequence Synthesized 482 Ser Trp Tyr Gly Gly Asp Ile Cys Leu Pro Trp Gly Cys Leu Trp Ser 1 5 10 15 Glu Glu Ser 483 18 PRT Artificial Sequence Synthesized 483 Thr Leu Phe Gln Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Glu Ser 484 18 PRT Artificial Sequence Synthesized 484 Val Gly Glu Trp Asp Ile Cys Leu Pro Asn Trp Gly Cys Leu Trp Glu 1 5 10 15 Arg Glu 485 18 PRT Artificial Sequence Synthesized 485 Trp Phe Pro Lys Asp Arg Cys Leu Pro Val Trp Gly Cys Leu Trp Glu 1 5 10 15 Arg His 486 20 PRT Artificial Sequence Synthesized 486 Trp Gly Met Ala Arg Asp Trp Cys Leu Pro Met Trp Gly Cys Leu Trp 1 5 10 15 Arg Gly Gly Gly 20 487 20 PRT Artificial Sequence Synthesized 487 Trp His Leu Thr Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Gly Asp Glu Gln 20 488 18 PRT Artificial Sequence Synthesized 488 Trp Trp Met Ala Asp Arg Cys Leu Pro Leu Trp Gly Cys Leu Trp Arg 1 5 10 15 Gly Asp 489 18 PRT Artificial Sequence Synthesized 489 Trp Trp Val Arg Asp Leu Cys Leu Pro Thr Trp Gly Cys Leu Trp Ser 1 5 10 15 Gly Lys 490 18 PRT Artificial Sequence Synthesized 490 Tyr Phe Asp Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly 1 5 10 15 Ser Asp 491 15 PRT Artificial Sequence Synthesized 491 Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp Asp 1 5 10 15 492 19 PRT Artificial Sequence synthesized 492 Ser Trp Tyr Gly Gly Asp Ile Cys Leu Pro Trp Gly Cys Leu Trp Ser 1 5 10 15 Glu Glu Ser 

1. A conjugate for prolonging the elimination half time of a therapeutic or diagnostic substance, comprising a peptide having an engineered amino acid sequence capable of binding a plasma protein, said peptide conjugated to the therapeutic or diagnostic substance, wherein the elimination half time of the conjugate exceeds that of the unconjugated therapeutic or diagnostic substance.
 2. The conjugate of claim 1, wherein the sequence is selected by screening a peptide library.
 3. The conjugate of claim 2, wherein the library screening comprises phage display of the peptides.
 4. The conjugate of claim 1, wherein the peptide is shorter than about 40 residues.
 5. The conjugate of claim 4, wherein the peptide is shorter than about 30 residues.
 6. The conjugate of claim 5, wherein the peptide is shorter than about 20 residues.
 7. The conjugate of claim 1, wherein the sequence is Xaa_(i)-Cys-Xaa_(j)-Cys-Xaa_(k), wherein the sum of i, j, and k is less than or equal to about
 25. 8. The conjugate of claim 1, wherein the plasma protein is an immunoglobulin.
 9. The conjugate of claim 8, wherein the immunoglobulin is IgG.
 10. The conjugate of claim 9, wherein the sequence is Xaa_(i)-Cys-Xaa_(j)-Cys-Xaa_(k), wherein the sum of i, j, and k is less than or equal to about
 25. 11. The conjugate of claim 10, wherein the sum of i, j, and k is less than or equal to about
 18. 12. The conjugate of claim 11, wherein the sum of i, j, and k is less than or equal to about
 11. 13. The conjugate of claim 8, wherein the immunoglobulin is IgM.
 14. The conjugate of claim 1, wherein the plasma protein is serum albumin.
 15. The conjugate of claim 1, wherein the therapeutic or diagnostic substance comprises a protein.
 16. The conjugate of claim 1, wherein the affinity of the peptide for the plasma protein is characterized by an equilibrium dissociation constant, K_(d), less than or equal to about 500 nM.
 17. The conjugate of claim 15, wherein K_(d) is less than or equal to about 100 nM.
 18. The conjugate of claim 17, wherein K_(d) is less than or equal to about 50 nM.
 19. The conjugate of claim 15, wherein the peptide is conjugated to the amino terminus of the protein.
 20. The conjugate of claim 19, further comprising a linker between the peptide and the protein.
 21. The conjugate of claim 15, wherein the peptide is conjugated to the carboxy terminus of the protein.
 22. The conjugate of claim 21, further comprising a linker between the peptide and the protein.
 23. The conjugate of claim 15, wherein the peptide is conjugated to a region of the protein other than the amino or carboxy terminus.
 24. A polynucleotide encoding the conjugate of claim
 19. 25. A polynucleotide encoding the conjugate of claim
 20. 26. A polynucleotide encoding the conjugate of claim
 21. 27. A polynucleotide encoding the conjugate of claim
 22. 28. A polynucleotide encoding the conjugate of claim
 23. 29. The conjugate of claim 1, wherein the peptide sequence is selected from the group consisting of SEQ ID NOS.: 2, 3, 8 and 17 through
 111. 30. The conjugate of claim 29, wherein the peptide sequence is SEQ ID NO:
 8. 31. A method of prolonging the elimination half time of a therapeutic substance, comprising conjugating the substance to a peptide having an engineered amino acid sequence capable of binding a plasma protein, wherein the elimination half time of the conjugated substance exceeds that of the unconjugated therapeutic substance.
 32. A peptide which: i) comprises the sequence Trp₁-Glu₁-Ala₁-Asp₁-Cys₁-Ala₂-Trp₂-His-Leu₁-Gly-Glu₂-Leu₂-Val-Trp₃-Cys₂-Thr-Pro-Met-Glu₃-Phe (SEQ ID NO: 16); or ii) competes with SEQ ID NO: 18 for binding IgG-Fc in an in vitro assay, and has between 1 and 6 amino acids of SEQ ID NO: 18 substituted according to the following: Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Gly-Glu-Leu-Val-Trp-Cys-Xaa₉-Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃ (SEQ ID NO: 14), wherein Xaa₄ is Ser, Arg, or Asp; Xaa₅ is Ala, Ser, or Thr; Xaa₆ is Trp, Tyr; Xaa₇ is His, Trp; Xaa₈ is Leu or Met; and Xaa₉ is Glu, Ser, Thr or Val; and iii) comprises the peptide of ii).
 33. The peptide of claim 32 having the following formula: Xaa_(i)-Cys₁-Xaa_(j)-Cys₂-Xaa_(k), wherein Xaa_(i) is absent or is between 1 and 4 amino acids; Xaa_(j) is 9 amino acids, and Xaa_(k) is absent or between 1 and 5 amino acids.
 34. The peptide of claim 32 having an IC₅₀ for IgG-Fc of less than about 1 îM.
 35. The peptide of claim 34 having an IC₅₀ for IgG-Fc of less than about 100 nM.
 36. The peptide of claim 35 having an IC₅₀ for IgG-Fc of less than about 10 nM.
 37. An isolated polynucleotide encoding the peptide of claim
 32. 38. The complement of an isolated polynucleotide encoding the peptide of claim
 32. 39. A peptide having between 11 and 20 amino acids which competes for binding to IgG-Fc in an in vitro assay with Trp₁-Glu₁-Ala₁-Asp-Cys₁-Ala₂-Trp₂-His-Leu₁-Gly-Glu₂-Leu₂-Val-Trp₃-Cys₂-Thr-Pro-Met-Glu₃-Phe (SEQ ID NO: 16) and having the formula Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Gly-Glu-Leu-Val-Trp-Cys-Xaa₉-Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃ (SEQ ID NO: 14), wherein Xaa₍₁₋₄₎ is absent or between one and four amino acids; Xaa₅ is selected from the group consisting of Ala, Ser, or Thr; Xaa₆ is selected from the group consisting of Trp or Tyr; Xaa₇ is selected from the group consisting of His or Trp; and Xaa₈ is selected from the group consisting of Leu or Met; and Xaa₍₉₋₁₃₎ is between one and five amino acids.
 40. An isolated polynucleotide encoding the peptide of claim
 39. 41. The complement of an isolated polynucleotide encoding the peptide of claim
 39. 42. A peptide conjugated to a therapeutic or diagnostic substance, wherein said peptide has an engineered amino acid sequence capable of binding a plasma protein, and wherein the elimination half time of the conjugate exceeds that of the unconjugated therapeutic or diagnostic substance.
 43. A peptide which: i) comprises the sequence (Xaa)₅-Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp-(Xaa)₄ (SEQ ID NO: 366) wherein Xaa is an amino acid or ii) competes for binding human serum albumin in an in vitro assay with a peptide having the sequence (Xaa)₅-Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp-(Xaa)₄ (SEQ ID NO: 366) wherein Xaa is an amino acid or iii) comprises the sequence of ii).
 44. The conjugate of claim 1 wherein the peptide sequence is the peptide of claim
 43. 45. A peptide having the sequence Asp-Leu-Cys-Leu-Arg-Asp-Trp-Gly-Cys-Leu-Trp (SEQ ID NO: 119).
 46. A peptide having the sequence Asp-Be-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp (SEQ ID NO: 120).
 47. A peptide having the sequence Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp (SEQ ID NO: 121).
 48. A peptide having the sequence Gln-Arg-Leu-Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-Asp-Phe (SEQ ID NO: 122).
 49. A peptide having the sequence Gln-Gly-Leu-Ile-Gly-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Gly-Asp-Ser-Val (SEQ ID NO: 123).
 50. A peptide having the sequence Gln-Gly-Leu-Ile-Gly-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Gly-Asp-Ser-Val-Lys (SEQ ID NO: 124).
 51. A peptide having the sequence Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-Asp (SEQ ID NO: 125).
 52. A peptide having the sequence Arg-Leu-Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-Asp (SEQ ID NO: 126).
 53. A peptide having the sequence Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-Asp (SEQ ID NO: 127).
 54. A peptide having the sequence Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp (SEQ ID NO: 121).
 55. A peptide having the sequence Arg-Leu-Met-Glu-Asp-Ile-Cys-Leu-Ala-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-Asp (SEQ ID NO: 128).
 56. The conjugate of claim 1 wherein the peptide sequence is the peptide of any of claims 44-55.
 57. A peptide which: i) comprises the sequence Phe-Cys-Xaa-Asp-Trp-Pro-Xaa-Xaa-Xaa-Ser-Cys (SEQ ID NO: 113) wherein Xaa is an amino acid or ii) competes for binding human serum albumin in an in vitro assay with a peptide comprising the sequence Phe-Cys-Xaa-Asp-Trp-Pro-Xaa-Xaa-Xaa-Ser-Cys (SEQ ID NO: 113) wherein Xaa is an amino acid or iii) comprises the sequence of ii).
 58. A peptide which: i) comprises the sequence Val-Cys-Tyr-Xaa-Xaa-Xaa-Ile-Cys-Phe (SEQ ID NO: 114) wherein Xaa is an amino acid or ii) competes for binding human serum albumin in an in vitro assay with a peptide having the sequence Val-Cys-Tyr-Xaa-Xaa-Xaa-Ile-Cys-Phe (SEQ ID NO: 114) wherein Xaa is an amino acid or iii) comprises the sequence of ii).
 59. A peptide which: i) comprises the sequence Cys-Tyr-Xaa₁-Pro-Gly-Xaa-Cys (SEQ ID NO: 115) wherein Xaa is an amino acid and Xaa₁ is selected from the group consisting of Ile, Phe, Tyr and Val or ii) competes for binding human serum albumin in an in vitro assay with a peptide having the sequence Cys-Tyr-Xaa₁-Pro-Gly-Xaa-Cys (SEQ ID NO: 115) wherein Xaa is an amino acid and Xaa₁ is selected from the group consisting of Ile, Phe, Tyr and Val or iii) comprises the sequence of ii).
 60. The conjugate of claim 1 wherein the peptide sequence is the peptide of any of claims 57, 58 or
 59. 